Abstract:

A transgenic plant transformed by a Protein Kinase Stress-Related Protein
(PKSRP) coding nucleic acid, wherein expression of the nucleic acid
sequence in the plant results in increased tolerance to environmental
stress as compared to a wild type variety of the plant. Also provided are
agricultural products, including seeds, produced by the transgenic
plants. Also provided are isolated PKSRPs, and isolated nucleic acid
coding PKSRPs, and vectors and host cells containing the latter.

Claims:

1. A transgenic plant cell transformed with an expression vector
comprising an isolated polynucleotide selected from the group consisting
of:a) a polynucleotide having a sequence comprising nucleotides 1 to 1441
of SEQ ID NO:17; andb) a polynucleotide encoding a polypeptide having a
sequence comprising amino acids 1 to 283 of SEQ ID NO:30.

2. The plant cell of claim 1, wherein the polynucleotide has the sequence
comprising nucleotides 1 to 1441 of SEQ ID NO:17.

3. The plant cell of claim 1, wherein the polynucleotide encodes the
polypeptide having the sequence comprising amino acids 1 to 283 of SEQ ID
NO:30.

4. A transgenic plant transformed with an expression cassette comprising
an isolated polynucleotide selected from the group consisting of:a) a
polynucleotide having a sequence comprising nucleotides 1 to 1441 of SEQ
ID NO17; andb) a polynucleotide encoding a polypeptide having a sequence
comprising amino acids 1 to 283 of SEQ ID NO:30.

5. The plant of claim 4, wherein the polynucleotide has the sequence
comprising nucleotides 1 to 1441 of SEQ ID NO:17.

6. The plant of claim 4, wherein the polynucleotide encodes the
polypeptide having the sequence comprising amino acids 1 to 283 of SEQ ID
NO:30.

14. A seed which is true breeding for a transgene comprising a
polynucleotide selected from the group consisting of:a) a polynucleotide
having a sequence comprising nucleotides 1 to 1441 of SEQ ID NO:17; andb)
a polynucleotide encoding a polypeptide having a sequence comprising
amino acids 1 to 283 of SEQ ID NO:30.

15. The seed of claim 14, wherein the polynucleotide has the sequence
comprising nucleotides 1 to 1441 of SEQ ID NO:17.

16. The seed of claim 14, wherein the polynucleotide encodes the
polypeptide having the sequence comprising amino acids 1 to 283 of SEQ ID
NO:30.

17. An isolated nucleic acid comprising a polynucleotide selected from the
group consisting of:a) a polynucleotide having a sequence comprising
nucleotides 1 to 1441 of SEQ ID NO:17; andb) a polynucleotide encoding a
polypeptide having a sequence comprising amino acids 1 to 283 of SEQ ID
NO:30.

18. The isolated nucleic acid of claim 17, wherein the polynucleotide has
the sequence comprising nucleotides 1 to 1441 of SEQ ID NO:17.

20. A method of producing a drought-tolerant transgenic plant, the method
comprising the steps of:a) transforming a plant cell with an expression
vector comprising a polynucleotide selected from the group consisting
of:a) a polynucleotide having a sequence comprising nucleotides 1 to 1441
of SEQ ID NO:17; andb) a polynucleotide encoding a polypeptide having a
sequence comprising amino acids 1 to 283 of SEQ ID NO:30.

21. The method of claim 20, wherein the polynucleotide has the sequence
comprising nucleotides 1 to 1441 of SEQ ID NO:17.

22. The method of claim 20, wherein the polynucleotide encodes the
polypeptide having the sequence comprising amino acids 1 to 283 of SEQ ID
NO:30.

Description:

CROSS REFERENCE TO RELATED APPLICATIONS

[0001]This application is a divisional of U.S. patent application Ser. No.
09/828,313, filed Apr. 6, 2001 and now U.S. Pat. No. 6,867,351, and is
copending with U.S. patent application Ser. No. 12/401,635, filed Mar.
11, 2009, which is copending with U.S. patent application Ser. No.
11/961,634, filed Dec. 20, 2007, and now U.S. Pat. No. 7,521,598, which
is a continuation of U.S. patent application Ser. No. 11/564,902, filed
Nov. 30, 2006, and now U.S. Pat. No. 7,504,559, which is a continuation
of U.S. patent application Ser. No. 10/768,863, filed Jan. 30, 2004 and
now U.S. Pat. No. 7,179,962, which is a divisional of U.S. patent
application Ser. No. 09/828,313, which claims the priority benefit of
U.S. Provisional Application Ser. No. 60/196,001 filed Apr. 7, 2000. The
contents of each of the above-identified applications are hereby
incorporated by reference.

[0005]Abiotic environmental stresses, such as drought stress, salinity
stress, heat stress, and cold stress, are major limiting factors of plant
growth and productivity. Crop losses and crop yield losses of major crops
such as rice, maize (corn) and wheat caused by these stresses represent a
significant economic and political factor and contribute to food
shortages in many underdeveloped countries.

[0006]Plants are typically exposed during their life cycle to conditions
of reduced environmental water content. Most plants have evolved
strategies to protect themselves against these conditions of desiccation.
However, if the severity and duration of the drought conditions are too
great, the effects on plant development, growth and yield of most crop
plants are profound. Furthermore, most of the crop plants are very
susceptible to higher salt concentrations in the soil. Continuous
exposure to drought and high salt causes major alterations in the plant
metabolism. These great changes in metabolism ultimately lead to cell
death and consequently yield losses.

[0007]Developing stress-tolerant plants is a strategy that has the
potential to solve or mediate at least some of these problems. However,
traditional plant breeding strategies to develop new lines of plants that
exhibit resistance (tolerance) to these types of stresses are relatively
slow and require specific resistant lines for crossing with the desired
line. Limited germplasm resources for stress tolerance and
incompatibility in crosses between distantly related plant species
represent significant problems encountered in conventional breeding.
Additionally, the cellular processes leading to drought, cold and salt
tolerance in model, drought- and/or salt-tolerant plants are complex in
nature and involve multiple mechanisms of cellular adaptation and
numerous metabolic pathways. This multi-component nature of stress
tolerance has not only made breeding for tolerance largely unsuccessful,
but has also limited the ability to genetically engineer stress tolerance
plants using biotechnological methods.

[0008]Drought, cold as well as salt stresses have a common theme important
for plant growth and that is water availability. Plants are exposed
during their entire life cycle to conditions of reduced environmental
water content. Most plants have evolved strategies to protect themselves
against these conditions of desiccation. However, if the severity and
duration of the drought conditions are too great, the effects on plant
development, growth and yield of most crop plants are profound. Since
high salt content in some soils result in less available water for cell
intake, its effect is similar to those observed under drought conditions.
Additionally, under freezing temperatures, plant cells loose water as a
result of ice formation that starts in the apoplast and withdraws water
from the symplast. Commonly, a plant's molecular response mechanisms to
each of these stress conditions are common and protein kinases play an
essential role in these molecular mechanisms.

[0009]Protein kinases represent a super family and the members of this
family catalyze the reversible transfer of a phosphate group of ATP to
serine, threonine and tyrosine amino acid side chains on target proteins.
Protein kinases are primary elements in signaling processes in plants and
have been reported to play crucial roles in perception and transduction
of signals that allow a cell (and the plant) to respond to environmental
stimuli. In particular, receptor protein kinases (RPKs) represent one
group of protein kinases that activate a complex array of intracellular
signaling pathways in response to the extracellular environment (Van der
Gear et al., 1994 Annu. Rev. Cell Biol. 10:251-337). RPKs are single-pass
transmembrane proteins that contain an amino-terminal signal sequence,
extracellular domains unique to each receptor, and a cytoplasmic kinase
domain. Ligand binding induces homo- or hetero-dimerization of RPKs, and
the resultant close proximity of the cytoplasmic domains results in
kinase activation by transphosphorylation. Although plants have many
proteins similar to RPKs, no ligand has been identified for these
receptor-like kinases (RLKs). The majority of plant RLKs that have been
identified belong to the family of Serine/Threonine (Ser/Thr) kinases,
and most have extracellular Leucine-rich repeats (Becraft, P W. 1998
Trends Plant Sci. 3:384-388).

[0011]Another type of signaling mechanism involves members of the
conserved SNF1 Serine/Threonine protein kinase family. These kinases play
essential roles in eukaryotic glucose and stress signaling. Plant
SNF1-like kinases participate in the control of key metabolic enzymes,
including HMGR, nitrate reductase, sucrose synthase, and sucrose
phosphate synthase (SPS). Genetic and biochemical data indicate that
sugar-dependent regulation of SNF1 kinases involves several other sensory
and signaling components in yeast, plants and animals.

[0012]Additionally, members of the Mitogen-Activated Protein Kinase (MAPK)
family have been implicated in the actions of numerous environmental
stresses in animals, yeasts and plants. It has been demonstrated that
both MAPK-like kinase activity and mRNA levels of the components of MAPK
cascades increase in response to environmental stress and plant hormone
signal transduction. MAP kinases are components of sequential kinase
cascades, which are activated by phosphorylation of threonine and
tyrosine residues by intermediate upstream MAP kinase kinases (MAPKKs).
The MAPKKs are themselves activated by phosphorylation of serine and
threonine residues by upstream kinases (MAPKKKs). A number of MAP Kinase
genes have been reported in higher plants.

[0014]The invention provides in some embodiments that the PKSRP and coding
nucleic acid are that found in members of the genus Physcomitrella. In
another preferred embodiment, the nucleic acid and protein are from a
Physcomitrella patens. The invention provides that the environmental
stress can be salinity, drought, temperature, metal, chemical, pathogenic
and oxidative stresses, or combinations thereof. In preferred
embodiments, the environmental stress can be drought or cold temperature.

[0015]The invention further provides a seed produced by a transgenic plant
transformed by a PKSRP coding nucleic acid, wherein the plant is true
breeding for increased tolerance to environmental stress as compared to a
wild type variety of the plant. The invention further provides a seed
produced by a transgenic plant expressing a PKSRP, wherein the plant is
true breeding for increased tolerance to environmental stress as compared
to a wild type variety of the plant.

[0016]The invention further provides an agricultural product produced by
any of the below-described transgenic plants, plant parts or seeds. The
invention further provides an isolated PKSRP as described below. The
invention further provides an isolated PKSRP coding nucleic acid, wherein
the PKSRP coding nucleic acid codes for a PKSRP as described below.

[0017]The invention further provides an isolated recombinant expression
vector comprising a PKSRP coding nucleic acid as described below, wherein
expression of the vector in a host cell results in increased tolerance to
environmental stress as compared to a wild type variety of the host cell.
The invention further provides a host cell containing the vector and a
plant containing the host cell.

[0018]The invention further provides a method of producing a transgenic
plant with a PKSRP coding nucleic acid, wherein expression of the nucleic
acid in the plant results in increased tolerance to environmental stress
as compared to a wild type variety of the plant comprising: (a)
transforming a plant cell with an expression vector comprising a PKSRP
coding nucleic acid, and (b) generating from the plant cell a transgenic
plant with an increased tolerance to environmental stress as compared to
a wild type variety of the plant. In preferred embodiments, the PKSRP and
PKSRP coding nucleic acid are as described below.

[0019]The present invention further provides a method of identifying a
novel PKSRP, comprising (a) raising a specific antibody response to a
PKSRP, or fragment thereof, as described below; (b) screening putative
PKSRP material with the antibody, wherein specific binding of the
antibody to the material indicates the presence of a potentially novel
PKSRP; and (c) identifying from the bound material a novel PKSRP in
comparison to known PKSRP. Alternatively, hybridization with nucleic acid
probes as described below can be used to identify novel PKSRP nucleic
acids.

[0020]The present invention also provides methods of modifying stress
tolerance of a plant comprising, modifying the expression of a PKSRP
nucleic acid in the plant, wherein the PKSRP is as described below. The
invention provides that this method can be performed such that the stress
tolerance is either increased or decreased. Preferably, stress tolerance
is increased in a plant via increasing expression of a PKSRP nucleic
acid.

[0039]The present invention may be understood more readily by reference to
the following detailed description of the preferred embodiments of the
invention and the Examples included herein. However, before the present
compounds, compositions, and methods are disclosed and described, it is
to be understood that this invention is not limited to specific nucleic
acids, specific polypeptides, specific cell types, specific host cells,
specific conditions, or specific methods, etc., as such may, of course,
vary, and the numerous modifications and variations therein will be
apparent to those skilled in the art. It is also to be understood that
the terminology used herein is for the purpose of describing specific
embodiments only and is not intended to be limiting. In particular, the
designation of the amino acid sequences as protein "Protein Kinase
Stress-Related Proteins" (PKSRPs), in no way limits the functionality of
those sequences.

[0040]The present invention provides a transgenic plant cell transformed
by a PKSRP coding nucleic acid, wherein expression of the nucleic acid
sequence in the plant cell results in increased tolerance to
environmental stress as compared to a wild type variety of the plant
cell. The invention further provides transgenic plant parts and
transgenic plants containing the plant cells described herein. Also
provided is a plant seed produced by a transgenic plant transformed by a
PKSRP coding nucleic acid, wherein the seed contains the PKSRP coding
nucleic acid, and wherein the plant is true breeding for increased
tolerance to environmental stress as compared to a wild type variety of
the plant. The invention further provides a seed produced by a transgenic
plant expressing a PKSRP, wherein the seed contains the PKSRP, and
wherein the plant is true breeding for increased tolerance to
environmental stress as compared to a wild type variety of the plant. The
invention also provides an agricultural product produced by any of the
below-described transgenic plants, plant parts and plant seeds.

[0041]As used herein, the term "variety" refers to a group of plants
within a species that share constant characters that separate them from
the typical form and from other possible varieties within that species.
While possessing at least one distinctive trait, a variety is also
characterized by some variation between individuals within the variety,
based primarily on the Mendelian segregation of traits among the progeny
of succeeding generations. A variety is considered "true breeding" for a
particular trait if it is genetically homozygous for that trait to the
extent that, when the true-breeding variety is self-pollinated, a
significant amount of independent segregation of the trait among the
progeny is not observed. In the present invention, the trait arises from
the transgenic expression of one or more DNA sequences introduced into a
plant variety.

[0043]The PKSRPs of the present invention are preferably produced by
recombinant DNA techniques. For example, a nucleic acid molecule encoding
the protein is cloned into an expression vector (as described below), the
expression vector is introduced into a host cell (as described below) and
the PKSRP is expressed in the host cell. The PKSRP can then be isolated
from the cells by an appropriate purification scheme using standard
protein purification techniques. Alternative to recombinant expression, a
PKSRP polypeptide, or peptide can be synthesized chemically using
standard peptide synthesis techniques. Moreover, native PKSRP can be
isolated from cells (e.g., Physcomitrella patens), for example using an
anti-PKSRP antibody, which can be produced by standard techniques
utilizing a PKSRP or fragment thereof.

[0045]As used herein, the term "environmental stress" refers to any
sub-optimal growing condition and includes, but is not limited to,
sub-optimal conditions associated with salinity, drought, temperature,
metal, chemical, pathogenic and oxidative stresses, or combinations
thereof. In preferred embodiments, the environmental stress can be
salinity, drought, or temperature, or combinations thereof, and in
particular, can be high salinity, low water content or low temperature.
It is also to be understood that as used in the specification and in the
claims, "a" or "an" can mean one or more, depending upon the context in
which it is used. Thus, for example, reference to "a cell" can mean that
at least one cell can be utilized.

[0046]As also used herein, the terms "nucleic acid" and "nucleic acid
molecule" are intended to include DNA molecules (e.g., cDNA or genomic
DNA) and RNA molecules (e.g., mRNA) and analogs of the DNA or RNA
generated using nucleotide analogs. This term also encompasses
untranslated sequence located at both the 3' and 5' ends of the coding
region of the gene: at least about 1000 nucleotides of sequence upstream
from the 5' end of the coding region and at least about 200 nucleotides
of sequence downstream from the 3' end of the coding region of the gene.
The nucleic acid molecule can be single-stranded or double-stranded, but
preferably is double-stranded DNA.

[0047]An "isolated" nucleic acid molecule is one that is substantially
separated from other nucleic acid molecules which are present in the
natural source of the nucleic acid. Preferably, an "isolated" nucleic
acid is free of some of the sequences which naturally flank the nucleic
acid (i.e., sequences located at the 5' and 3' ends of the nucleic acid)
in the genomic DNA of the organism from which the nucleic acid is
derived. For example, in various embodiments, the isolated PKSRP nucleic
acid molecule can contain less than about 5 kb, 4 kb, 3 kb, 2 kb, 1 kb,
0.5 kb or 0.1 kb of nucleotide sequences which naturally flank the
nucleic acid molecule in genomic DNA of the cell from which the nucleic
acid is derived (e.g., a Physcomitrella patens cell). Moreover, an
"isolated" nucleic acid molecule, such as a cDNA molecule, can be free
from some of the other cellular material with which it is naturally
associated, or culture medium when produced by recombinant techniques, or
chemical precursors or other chemicals when chemically synthesized.

[0050]Moreover, the nucleic acid molecule of the invention can comprise
only a portion of the coding region of one of the sequences in SEQ ID
NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID
NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID
NO:24, SEQ ID NO:25 and SEQ ID NO:26, for example, a fragment which can
be used as a probe or primer or a fragment encoding a biologically active
portion of a PKSRP. The nucleotide sequences determined from the cloning
of the PKSRP genes from P. patens allow for the generation of probes and
primers designed for use in identifying and/or cloning PKSRP homologs in
other cell types and organisms, as well as PKSRP homologs from other
mosses and related species.

[0051]Portions of proteins encoded by the PKSRP nucleic acid molecules of
the invention are preferably biologically active portions of one of the
PKSRPs described herein. As used herein, the term "biologically active
portion of" a PKSRP is intended to include a portion, e.g., a
domain/motif, of a PKSRP that participates in a stress tolerance response
in a plant, has an activity as set forth in Table 1, or participates in
the transcription of a protein involved in a stress tolerance response in
a plant. To determine whether a PKSRP, or a biologically active portion
thereof, can participate in transcription of a protein involved in a
stress tolerance response in a plant, or whether repression of a PKSRP
results in increased stress tolerance in a plant, a stress analysis of a
plant comprising the PKSRP may be performed. Such analysis methods are
well known to those skilled in the art, as detailed in Example 7. More
specifically, nucleic acid fragments encoding biologically active
portions of a PKSRP can be prepared by isolating a portion of one of the
sequences in SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ
ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID
NO:36, SEQ ID NO:37, SEQ ID NO:38 and SEQ ID NO:39, expressing the
encoded portion of the PKSRP or peptide (e.g., by recombinant expression
in vitro) and assessing the activity of the encoded portion of the PKSRP
or peptide.

[0052]Biologically active portions of a PKSRP are encompassed by the
present invention and include peptides comprising amino acid sequences
derived from the amino acid sequence of a PKSRP, e.g., an amino acid
sequence of SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ
ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID
NO:36, SEQ ID NO:37, SEQ ID NO:38 or SEQ ID NO:39, or the amino acid
sequence of a protein homologous to a PKSRP, which include fewer amino
acids than a full length PKSRP or the full length protein which is
homologous to a PKSRP, and exhibit at least one activity of a PKSRP.
Typically, biologically active portions (e.g., peptides which are, for
example, 5, 10, 15, 20, 30, 35, 36, 37, 38, 39, 40, 50, 100 or more amino
acids in length) comprise a domain or motif with at least one activity of
a PKSRP. Moreover, other biologically active portions in which other
regions of the protein are deleted, can be prepared by recombinant
techniques and evaluated for one or more of the activities described
herein. Preferably, the biologically active portions of a PKSRP include
one or more selected domains/motifs or portions thereof having biological
activity.

[0053]The invention also provides PKSRP chimeric or fusion proteins. As
used herein, a PKSRP "chimeric protein" or "fusion protein" comprises a
PKSRP polypeptide operatively linked to a non-PKSRP polypeptide. A PKSRP
polypeptide refers to a polypeptide having an amino acid sequence
corresponding to a PKSRP, whereas a non-PKSRP polypeptide refers to a
polypeptide having an amino acid sequence corresponding to a protein
which is not substantially homologous to the PKSRP, e.g., a protein that
is different from the PKSRP and is derived from the same or a different
organism. Within the fusion protein, the term "operatively linked" is
intended to indicate that the PKSRP polypeptide and the non-PKSRP
polypeptide are fused to each other so that both sequences fulfill the
proposed function attributed to the sequence used. The non-PKSRP
polypeptide can be fused to the N-terminus or C-terminus of the PKSRP
polypeptide. For example, in one embodiment, the fusion protein is a
GST-PKSRP fusion protein in which the PKSRP sequences are fused to the
C-terminus of the GST sequences. Such fusion proteins can facilitate the
purification of recombinant PKSRPs. In another embodiment, the fusion
protein is a PKSRP containing a heterologous signal sequence at its
N-terminus. In certain host cells (e.g., mammalian host cells),
expression and/or secretion of a PKSRP can be increased through use of a
heterologous signal sequence.

[0054]Preferably, a PKSRP chimeric or fusion protein of the invention is
produced by standard recombinant DNA techniques. For example, DNA
fragments coding for the different polypeptide sequences are ligated
together in-frame in accordance with conventional techniques, for example
by employing blunt-ended or stagger-ended termini for ligation,
restriction enzyme digestion to provide for appropriate termini,
filling-in of cohesive ends as appropriate, alkaline phosphatase
treatment to avoid undesirable joining and enzymatic ligation. In another
embodiment, the fusion gene can be synthesized by conventional techniques
including automated DNA synthesizers. Alternatively, PCR amplification of
gene fragments can be carried out using anchor primers which give rise to
complementary overhangs between two consecutive gene fragments which can
subsequently be annealed and re-amplified to generate a chimeric gene
sequence (see, for example, Current Protocols in Molecular Biology, Eds.
Ausubel et al. John Wiley & Sons: 1992). Moreover, many expression
vectors are commercially available that already encode a fusion moiety
(e.g., a GST polypeptide), A PKSRP encoding nucleic acid can be cloned
into such an expression vector such that the fusion moiety is linked
in-frame to the PKSRP.

[0056]An agonist of the PKSRP can retain substantially the same, or a
subset, of the biological activities of the PKSRP. An antagonist of the
PKSRP can inhibit one or more of the activities of the naturally
occurring form of the PKSRP. For example, the PKSRP antagonist can
competitively bind to a downstream or upstream member of the cell
membrane component metabolic cascade that includes the PKSRP, or bind to
a PKSRP that mediates transport of compounds across such membranes,
thereby preventing translocation from taking place.

[0057]Nucleic acid molecules corresponding to natural allelic variants and
analogs, orthologs and paralogs of a PKSRP cDNA can be isolated based on
their identity to the Physcomitrella patens PKSRP nucleic acids described
herein using PKSRP cDNAs, or a portion thereof, as a hybridization probe
according to standard hybridization techniques under stringent
hybridization conditions. In an alternative embodiment, homologs of the
PKSRP can be identified by screening combinatorial libraries of mutants,
e.g., truncation mutants, of the PKSRP for PKSRP agonist or antagonist
activity. In one embodiment, a variegated library of PKSRP variants is
generated by combinatorial mutagenesis at the nucleic acid level and is
encoded by a variegated gene library. A variegated library of PKSRP
variants can be produced by, for example, enzymatically ligating a
mixture of synthetic oligonucleotides into gene sequences such that a
degenerate set of potential PKSRP sequences is expressible as individual
polypeptides, or alternatively, as a set of larger fusion proteins (e.g.,
for phage display) containing the set of PKSRP sequences therein. There
are a variety of methods that can be used to produce libraries of
potential PKSRP homologs from a degenerate oligonucleotide sequence.
Chemical synthesis of a degenerate gene sequence can be performed in an
automatic DNA synthesizer, and the synthetic gene is then ligated into an
appropriate expression vector. Use of a degenerate set of genes allows
for the provision, in one mixture, of all of the sequences encoding the
desired set of potential PKSRP sequences. Methods for synthesizing
degenerate oligonucleotides are known in the art (see, e.g., Narang, S.
A., 1983 Tetrahedron 39:3; Itakura et al., 1984 Annu. Rev. Biochem.
53:323; Itakura et al., 1984 Science 198:1056; Ike et al, 1983 Nucleic
Acid Res. 11:477).

[0058]In addition, libraries of fragments of the PKSRP coding regions can
be used to generate a variegated population of PKSRP fragments for
screening and subsequent selection of homologs of a PKSRP. In one
embodiment, a library of coding sequence fragments can be generated by
treating a double stranded PCR fragment of a PKSRP coding sequence with a
nuclease under conditions wherein nicking occurs only about once per
molecule, denaturing the double stranded DNA, renaturing the DNA to form
double stranded DNA, which can include sense/antisense pairs from
different nicked products, removing single stranded portions from
reformed duplexes by treatment with S1 nuclease, and ligating the
resulting fragment library into an expression vector. By this method, an
expression library can be derived which encodes N-terminal, C-terminal
and internal fragments of various sizes of the PKSRP.

[0059]Several techniques are known in the art for screening gene products
of combinatorial libraries made by point mutations or truncation, and for
screening cDNA libraries for gene products having a selected property.
Such techniques are adaptable for rapid screening of the gene libraries
generated by the combinatorial mutagenesis of PKSRP homologs. The most
widely used techniques, which are amenable to high through-put analysis,
for screening large gene libraries typically include cloning the gene
library into replicable expression vectors, transforming appropriate
cells with the resulting library of vectors, and expressing the
combinatorial genes under conditions in which detection of a desired
activity facilitates isolation of the vector encoding the gene whose
product was detected. Recursive ensemble mutagenesis (REM), a new
technique that enhances the frequency of functional mutants in the
libraries, can be used in combination with the screening assays to
identify PKSRP homologs (Arkin and Yourvan, 1992 PNAS 89:7811-7815;
Delgrave et al., 1993 Protein Engineering 6(3):327-331). In another
embodiment, cell based assays can be exploited to analyze a variegated
PKSRP library, using methods well known in the art. The present invention
further provides a method of identifying a novel PKSRP, comprising (a)
raising a specific antibody response to a PKSRP, or a fragment thereof,
as described herein; (b) screening putative PKSRP material with the
antibody, wherein specific binding of the antibody to the material
indicates the presence of a potentially novel PKSRP; and (c) analyzing
the bound material in comparison to known PKSRP, to determine its
novelty.

[0062]In another preferred embodiment, an isolated nucleic acid molecule
of the invention comprises a nucleotide sequence which is at least about
50-60%, preferably at least about 60-70%, more preferably at least about
70-80%, 80-90%, or 90-95%, and even more preferably at least about 95%,
96%, 97%, 98%, 99% or more homologous to a nucleotide sequence shown in
SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ
ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID
NO:24, SEQ ID NO:25 or SEQ ID NO:26, or a portion thereof. The preferable
length of sequence comparison for nucleic acids is at least 75
nucleotides, more preferably at least 100 nucleotides and most preferably
the entire length of the coding region.

[0063]It is also preferable that the homologous nucleic acid molecule of
the invention encodes a protein or portion thereof which includes an
amino acid sequence which is sufficiently homologous to an amino acid
sequence of SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ
ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID
NO:36, SEQ ID NO:37, SEQ ID NO:38 or SEQ ID NO:39 such that the protein
or portion thereof maintains the same or a similar function as the amino
acid sequence to which it is compared. Functions of the PKSRP amino acid
sequences of the present invention include the ability to participate in
a stress tolerance response in a plant, or more particularly, to
participate in the transcription of a protein involved in a stress
tolerance response in a Physcomitrella patens plant. Examples of such
activities are described in Table 1.

[0064]In addition to the above described methods, a determination of the
percent homology between two sequences can be accomplished using a
mathematical algorithm. A preferred, non-limiting example of a
mathematical algorithm utilized for the comparison of two sequences is
the algorithm of Karlin and Altschul (1990 Proc. Natl. Acad. Sci. USA
90:5873-5877). Such an algorithm is incorporated into the NBLAST and
XBLAST programs of Altschul, et al. (1990 J. Mol. Biol. 215:403-410).

[0065]BLAST nucleic acid searches can be performed with the NBLAST
program, score=100, wordlength=12 to obtain nucleic acid sequences
homologous to the PKSRP nucleic acid molecules of the invention.
Additionally, BLAST protein searches can be performed with the XBLAST
program, score=50, wordlength=3 to obtain amino acid sequences homologous
to PKSRPs of the present invention. To obtain gapped alignments for
comparison purposes, Gapped BLAST can be utilized as described in
Altschul et al. (1997 Nucleic Acids Res. 25:3389-3402), When utilizing
BLAST and Gapped BLAST programs, the default parameters of the respective
programs (e.g., XBLAST and NBLAST) can be used. Another preferred
non-limiting example of a mathematical algorithm utilized for the
comparison of sequences is the algorithm of Myers and Miller (CABIOS
1989). Such an algorithm is incorporated into the ALIGN program (version
2.0) that is part of the GCG sequence alignment software package. When
utilizing the ALIGN program for comparing amino acid sequences, a PAM120
weight residue table, a gap length penalty of 12 and a gap penalty of 4
can be used to obtain amino acid sequences homologous to the PKSRPs of
the present invention. To obtain gapped alignments for comparison
purposes, Gapped BLAST can be utilized as described in Altschul et al.
(1997 Nucleic Acids Res. 25:3389-3402). When utilizing BLAST and Gapped
BLAST programs, the default parameters of the respective programs (e.g.,
XBLAST and NBLAST) can be used. Another preferred non-limiting example of
a mathematical algorithm utilized for the comparison of sequences is the
algorithm of Myers and Miller (CABIOS1989). Such an algorithm is
incorporated into the ALIGN program (version 2.0) that is part of the GCG
sequence alignment software package. When utilizing the ALIGN program for
comparing amino acid sequences, a PAM120 weight residue table, a gap
length penalty of 12 and a gap penalty of 4 can be used.

[0067]As used herein, the term "hybridizes under stringent conditions" is
intended to describe conditions for hybridization and washing under which
nucleotide sequences at least 60% homologous to each other typically
remain hybridized to each other. Preferably, the conditions are such that
sequences at least about 65%, more preferably at least about 70%, and
even more preferably at least about 75% or more homologous to each other
typically remain hybridized to each other. Such stringent conditions are
known to those skilled in the art and can be found in Current Protocols
in Molecular Biology, 6.3.1-6.3.6, John Wiley & Sons, N.Y. (1989). A
preferred, non-limiting example of stringent hybridization conditions are
hybridization in 6× sodium chloride/sodium citrate (SSC) at about
45° C., followed by one or more washes in 0.2×SSC, 0.1% SDS
at 50-65° C.; Preferably, an isolated nucleic acid molecule of the
invention that hybridizes under stringent conditions to a sequence of SEQ
ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID
NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID
NO:24, SEQ ID NO:25 or SEQ ID NO:26 corresponds to a naturally occurring
nucleic acid molecule. As used herein, a "naturally occurring" nucleic
acid molecule refers to an RNA or DNA molecule having a nucleotide
sequence that occurs in nature (e.g., encodes a natural protein). In one
embodiment, the nucleic acid encodes a naturally occurring Physcomitrella
patens PKSRP.

[0068]Using the above-described methods, and others known to those of
skill in the art, one of ordinary skill in the art can isolate homologs
of the PKSRPs comprising amino acid sequences shown in SEQ ID NO:27, SEQ
ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, SEQ ID
NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37, SEQ ID
NO:38 or SEQ ID NO:39. One subset of these homologs are allelic variants,
As used herein, the term "allelic variant" refers to a nucleotide
sequence containing polymorphisms that lead to changes in the amino acid
sequences of a PKSRP and that exist within a natural population (e.g., a
plant species or variety). Such natural allelic variations can typically
result in 1-5% variance in a PKSRP nucleic acid. Allelic variants can be
identified by sequencing the nucleic acid sequence of interest in a
number of different plants, which can be readily carried out by using
hybridization probes to identify the same PKSRP genetic locus in those
plants. Any and all such nucleic acid variations and resulting amino acid
polymorphisms or variations in a PKSRP that are the result of natural
allelic variation and that do not alter the functional activity of a
PKSRP, are intended to be within the scope of the invention.

[0069]Moreover, nucleic acid molecules encoding PKSRPs from the same or
other species such as PKSRP analogs, orthologs and paralogs, are intended
to be within the scope of the present invention. As used herein, the term
"analogs" refers to two nucleic acids that have the same or similar
function, but that have evolved separately in unrelated organisms. As
used herein, the term "orthologs" refers to two nucleic acids from
different species, but that have evolved from a common ancestral gene by
speciation. Normally, orthologs encode proteins having the same or
similar functions. As also used herein, the term "paralogs" refers to two
nucleic acids that are related by duplication within a genome. Paralogs
usually have different functions, but these functions may be related
(Tatusov, R. L, et al. 1997 Science 278(5338):631-637). Analogs,
orthologs and paralogs of a naturally occurring PKSRP can differ from the
naturally occurring PKSRP by post-translational modifications, by amino
acid sequence differences, or by both. Post-translational modifications
include in vivo and in vitro chemical derivatization of polypeptides,
e.g., acetylation, carboxylation, phosphorylation, or glycosylation, and
such modifications may occur during polypeptide synthesis or processing
or following treatment with isolated modifying enzymes. In particular,
orthologs of the invention will generally exhibit at least 80-85%, more
preferably 90%, and most preferably 95%, 96%, 97%, 98% or even 99%
identity or homology with all or part of a naturally occurring PKSRP
amino acid sequence and will exhibit a function similar to a PKSRP.
Orthologs of the present invention are also preferably capable of
participating in the stress response in plants. In one embodiment, the
PKSRP orthologs maintain the ability to participate in the metabolism of
compounds necessary for the construction of cellular membranes in
Physcomitrella patens, or in the transport of molecules across these
membranes.

[0070]In addition to naturally-occurring variants of a PKSRP sequence that
may exist in the population, the skilled artisan will further appreciate
that changes can be introduced by mutation into a nucleotide sequence of
SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ
ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID
NO:24, SEQ ID NO:25 or SEQ ID NO:26, thereby leading to changes in the
amino acid sequence of the encoded PKSRP, without altering the functional
ability of the PKSRP. For example, nucleotide substitutions leading to
amino acid substitutions at "non-essential" amino acid residues can be
made in a sequence of SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID
NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID
NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ED NO:25 or SEQ ID NO:26, A
"non-essential" amino acid residue is a residue that can be altered from
the wild-type sequence of one of the PKSRPs without altering the activity
of said PKSRP, whereas an "essential" amino acid residue is required for
PKSRP activity. Other amino acid residues, however, (e.g., those that are
not conserved or only semi-conserved in the domain having PKSRP activity)
may not be essential for activity and thus are likely to be amenable to
alteration without altering PKSRP activity.

[0073]Families of amino acid residues having similar side chains have been
defined in the art. These families include amino acids with basic side
chains (e.g., lysine, arginine, histidine), acidic side chains (e.g.,
aspartic acid, glutamic acid), uncharged polar side chains (e.g.,
glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine),
nonpolar side chains (e.g., alanine, valine, leucine, isoleucine,
proline, phenylalanine, methionine, tryptophan), beta-branched side
chains (e.g., threonine, valine, isoleucine) and aromatic side chains
(e.g., tyrosine, phenylalanine, tryptophan, histidine). Thus, a predicted
nonessential amino acid residue in a PKSRP is preferably replaced with
another amino acid residue from the same side chain family.
Alternatively, in another embodiment, mutations can be introduced
randomly along all or part of a PKSRP coding sequence, such as by
saturation mutagenesis, and the resultant mutants can be screened for a
PKSRP activity described herein to identify mutants that retain PKSRP
activity. Following mutagenesis of one of the sequences of SEQ ID NO:14,
SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ
ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID
NO:25 and SEQ ID NO:26, the encoded protein can be expressed
recombinantly and the activity of the protein can be determined by
analyzing the stress tolerance of a plant expressing the protein as
described in Example 7.

[0074]In addition to the nucleic acid molecules encoding the PKSRPs
described above, another aspect of the invention pertains to isolated
nucleic acid molecules that are antisense thereto. An "antisense" nucleic
acid comprises a nucleotide sequence that is complementary to a "sense"
nucleic acid encoding a protein, e.g., complementary to the coding strand
of a double-stranded cDNA molecule or complementary to an mRNA sequence.
Accordingly, an antisense nucleic acid can hydrogen bond to a sense
nucleic acid. The antisense nucleic acid can be complementary to an
entire PKSRP coding strand, or to only a portion thereof. In one
embodiment, an antisense nucleic acid molecule is antisense to a "coding
region" of the coding strand of a nucleotide sequence encoding a PKSRP.
The term "coding region" refers to the region of the nucleotide sequence
comprising codons that are translated into amino acid residues (e.g., the
entire coding region of , , , comprises nucleotides 1 to . . . ). In
another embodiment, the antisense nucleic acid molecule is antisense to a
"noncoding region" of the coding strand of a nucleotide sequence encoding
a PKSRP. The term "noncoding region" refers to 5' and 3' sequences that
flank the coding region that are not translated into amino acids (i.e.,
also referred to as 5' and 3' untranslated regions).

[0076]Given the coding strand sequences encoding the PKSRPs disclosed
herein (e.g., the sequences set forth in SEQ ID NO:14, SEQ ID NO:15, SEQ
ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID
NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25 and SEQ ID
NO:26), antisense nucleic acids of the invention can be designed
according to the rules of Watson and Crick base pairing. The antisense
nucleic acid molecule can be complementary to the entire coding region of
PKSRP mRNA, but more preferably is an oligonucleotide which is antisense
to only a portion of the coding or noncoding region of PKSRP mRNA. For
example, the antisense oligonucleotide can be complementary to the region
surrounding the translation start site of PKSRP mRNA. An antisense
oligonucleotide can be, for example, about 5, 10, 15, 20, 25, 30, 35, 40,
45 or 50 nucleotides in length.

[0077]An antisense nucleic acid of the invention can be constructed using
chemical synthesis and enzymatic ligation reactions using procedures
known in the art. For example, an antisense nucleic acid (e.g., an
antisense oligonucleotide) can be chemically synthesized using naturally
occurring nucleotides or variously modified nucleotides designed to
increase the biological stability of the molecules or to increase the
physical stability of the duplex formed between the antisense and sense
nucleic acids, e.g., phosphorothioate derivatives and acridine
substituted nucleotides can be used. Examples of modified nucleotides
which can be used to generate the antisense nucleic acid include
5-fluorouracil, 5-bromouracil, 5-chlorouracil, 5-iodouracil,
hypoxanthine, xanthine, 4-acetylcytosine, 5-(carboxyhydroxylmethyl)
uracil, 5-carboxymethylaminomethyl-2-thiouridine,
5-carboxymethylaminomethyluracil, dihydrouracil,
beta-D-galactosylqueosine, inosine, N6-isopentenyladenine,
1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine,
2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-adenine,
7-methylguanine, 5-methylaminomethyluracil,
5-methoxyaminomethyl-2-thiouracil, beta-D-mannosylqueosine,
5'-methoxycarboxymethyluracil, 5-methoxyuracil,
2-methylthio-N-6-isopentenyladenine, uracil-5-oxyacetic acid (v),
wybutoxosine, pseudouracil, queosine, 2-thiocytosine,
5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil,
uracil-5-oxyacetic acid methylester, uracil-5-oxyacetic acid (v),
5-methyl-2-thiouracil, 3-(3-amino-3-N-2-carboxypropyl) uracil, (acp3)w,
and 2,6-diaminopurine. Alternatively, the antisense nucleic acid can be
produced biologically using an expression vector into which a nucleic
acid has been subcloned in an antisense orientation (i.e., RNA
transcribed from the inserted nucleic acid will be of an antisense
orientation to a target nucleic acid of interest, described further in
the following subsection).

[0078]The antisense nucleic acid molecules of the invention are typically
administered to a cell or generated in situ such that they hybridize with
or bind to cellular mRNA and/or genomic DNA encoding a PKSRP to thereby
inhibit expression of the protein, e.g., by inhibiting transcription
and/or translation. The hybridization can be by conventional nucleotide
complementarity to form a stable duplex, or, for example, in the case of
an antisense nucleic acid molecule which binds to DNA duplexes, through
specific interactions in the major groove of the double helix. The
antisense molecule can be modified such that it specifically binds to a
receptor or an antigen expressed on a selected cell surface, e.g., by
linking the antisense nucleic acid molecule to a peptide or an antibody
which binds to a cell surface receptor or antigen. The antisense nucleic
acid molecule can also be delivered to cells using the vectors described
herein. To achieve sufficient intracellular concentrations of the
antisense molecules, vector constructs in which the antisense nucleic
acid molecule is placed under the control of a strong prokaryotic, viral,
or eukaryotic (including plant) promoter are preferred.

[0080]In still another embodiment, an antisense nucleic acid of the
invention is a ribozyme. Ribozymes are catalytic RNA molecules with
ribonuclease activity which are capable of cleaving a single-stranded
nucleic acid, such as an mRNA, to which they have a complementary region.
Thus, ribozymes (e.g., hammerhead ribozymes described in Haselhoff and
Gerlach, 1988 Nature 334:585-591) can be used to catalytically cleave
PKSRP mRNA transcripts to thereby inhibit translation of PKSRP mRNA. A
ribozyme having specificity for a PKSRP-encoding nucleic acid can be
designed based upon the nucleotide sequence of a PKSRP cDNA, as disclosed
herein (i.e., SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ
ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID
NO:23, SEQ ID NO:24, SEQ ID NO:25 or SEQ ID NO:26) or on the basis of a
heterologous sequence to be isolated according to methods taught in this
invention. For example, a derivative of a Tetrahymena L-19 IVS RNA can be
constructed in which the nucleotide sequence of the active site is
complementary to the nucleotide sequence to be cleaved in a
PKSRP-encoding mRNA. See, e.g., Cech et al. U.S. Pat. No. 4,987,071 and
Cech et al. U.S. Pat. No. 5,116,742. Alternatively, PKSRP mRNA can be
used to select a catalytic RNA having a specific ribonuclease activity
from a pool of RNA molecules. See, e.g., Bartel, D. and Szostak, J. W.,
1993 Science 261:1411-1418.

[0082]In addition to the PKSRP nucleic acids and proteins described above,
the present invention encompasses these nucleic acids and proteins
attached to a moiety. These moieties include, but are not limited to,
detection moieties, hybridization moieties, purification moieties,
delivery moieties, reaction moieties, binding moieties, and the like. A
typical group of nucleic acids having moieties attached are probes and
primers. The probes and primers typically comprise a substantially
isolated oligonucleotide. The oligonucleotide typically comprises a
region of nucleotide sequence that hybridizes under stringent conditions
to at least about 12, preferably about 25, more preferably about 40, 50
or 75 consecutive nucleotides of a sense strand of one of the sequences
set forth in SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ
ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID
NO:23, SEQ ID NO:24, SEQ ID NO:25 and SEQ ID NO:26, an anti-sense
sequence of one of the sequences set forth in SEQ ID NO:14, SEQ ID NO:15,
SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ
ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25 and SEQ
ID NO:26, or naturally occurring mutants thereof. Primers based on a
nucleotide sequence of SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID
NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID
NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25 or SEQ ID NO:26 can be
used in PCR reactions to clone PKSRP homologs. Probes based on the PKSRP
nucleotide sequences can be used to detect transcripts or genomic
sequences encoding the same or homologous proteins. In preferred
embodiments, the probe further comprises a label group attached thereto,
e.g. the label group can be a radioisotope, a fluorescent compound, an
enzyme, or an enzyme co-factor. Such probes can be used as a part of a
genomic marker test kit for identifying cells which express a PKSRP, such
as by measuring a level of a PKSRP-encoding nucleic acid, in a sample of
cells, e.g., detecting PKSRP mRNA levels or determining whether a genomic
PKSRP gene has been mutated or deleted.

[0083]In particular, a useful method to ascertain the level of
transcription of the gene (an indicator of the amount of mRNA available
for translation to the gene product) is to perform a Northern blot (for
reference see, for example, Ausubel et al., 1988 Current Protocols in
Molecular Biology, Wiley: New York). This information at least partially
demonstrates the degree of transcription of the transformed gene. Total
cellular RNA can be prepared from cells, tissues or organs by several
methods, all well-known in the art, such as that described in Bormann, E.
R. et al., 1992 Mol. Microbiol. 6:317-326. To assess the presence or
relative quantity of protein translated from this mRNA, standard
techniques, such as a Western blot, may be employed. These techniques are
well known to one of ordinary skill in the art. (See, for example,
Ausubel et al., 1988 Current Protocols in Molecular Biology, Wiley: New
York).

[0084]The invention further provides an isolated recombinant expression
vector comprising a PKSRP nucleic acid as described above, wherein
expression of the vector in a host cell results in increased tolerance to
environmental stress as compared to a wild type variety of the host cell.
As used herein, the term "vector" refers to a nucleic acid molecule
capable of transporting another nucleic acid to which it has been linked.
One type of vector is a "plasmid", which refers to a circular double
stranded DNA loop into which additional DNA segments can be ligated.
Another type of vector is a viral vector, wherein additional DNA segments
can be ligated into the viral genome. Certain vectors are capable of
autonomous replication in a host cell into which they are introduced
(e.g., bacterial vectors having a bacterial origin of replication and
episomal mammalian vectors). Other vectors (e.g., non-episomal mammalian
vectors) are integrated into the genome of a host cell upon introduction
into the host cell, and thereby are replicated along with the host
genome. Moreover, certain vectors are capable of directing the expression
of genes to which they are operatively linked. Such vectors are referred
to herein as "expression vectors". In general, expression vectors of
utility in recombinant DNA techniques are often in the form of plasmids.
In the present specification, "plasmid" and "vector" can be used
interchangeably as the plasmid is the most commonly used form of vector.
However, the invention is intended to include such other forms of
expression vectors, such as viral vectors (e.g., replication defective
retroviruses, adenoviruses and adeno-associated viruses), which serve
equivalent functions.

[0085]The recombinant expression vectors of the invention comprise a
nucleic acid of the invention in a form suitable for expression of the
nucleic acid in a host cell, which means that the recombinant expression
vectors include one or more regulatory sequences, selected on the basis
of the host cells to be used for expression, which is operatively linked
to the nucleic acid sequence to be expressed. Within a recombinant
expression vector, "operably linked" is intended to mean that the
nucleotide sequence of interest is linked to the regulatory sequence(s)
in a manner which allows for expression of the nucleotide sequence (e.g.,
in an in vitro transcription/translation system or in a host cell when
the vector is introduced into the host cell). The term "regulatory
sequence" is intended to include promoters, enhancers and other
expression control elements (e.g., polyadenylation signals). Such
regulatory sequences are described, for example, in Goeddel, Gene
Expression Technology: Methods in Enzymology 185, Academic Press, San
Diego, Calif. (1990) or see: Gruber and Crosby, in: Methods in Plant
Molecular Biology and Biotechnology, eds. Glick and Thompson, Chapter 7,
89-108, CRC Press: Boca Raton, Fla., including the references therein.
Regulatory sequences include those that direct constitutive expression of
a nucleotide sequence in many types of host cells and those that direct
expression of the nucleotide sequence only in certain host cells or under
certain conditions. It will be appreciated by those skilled in the art
that the design of the expression vector can depend on such factors as
the choice of the host cell to be transformed, the level of expression of
protein desired, etc. The expression vectors of the invention can be
introduced into host cells to thereby produce proteins or peptides,
including fusion proteins or peptides, encoded by nucleic acids as
described herein (e.g., PKSRPs, mutant forms of PKSRPs, fusion proteins,
etc.).

[0087]Expression of proteins in prokaryotes is most often carried out with
vectors containing constitutive or inducible promoters directing the
expression of either fusion or non-fusion proteins. Fusion vectors add a
number of amino acids to a protein encoded therein, usually to the amino
terminus of the recombinant protein but also to the C-terminus or fused
within suitable regions in the proteins. Such fusion vectors typically
serve three purposes: 1) to increase expression of a recombinant protein;
2) to increase the solubility of a recombinant protein; and 3) to aid in
the purification of a recombinant protein by acting as a ligand in
affinity purification. Often, in fusion expression vectors, a proteolytic
cleavage site is introduced at the junction of the fusion moiety and the
recombinant protein to enable separation of the recombinant protein from
the fusion moiety subsequent to purification of the fusion protein. Such
enzymes, and their cognate recognition sequences, include Factor Xa,
thrombin and enterokinase.

[0088]Typical fusion expression vectors include pGEX (Pharmacia Biotech
Inc; Smith, D. B. and Johnson, K. S., 1988 Gene 67:31-40), pMAL (New
England Biolabs, Beverly, Mass.) and pRIT5 (Pharmacia, Piscataway, N.J.)
which fuse glutathione S-transferase (GST), maltose E binding protein, or
protein A, respectively, to the target recombinant protein. In one
embodiment, the coding sequence of the PKSRP is cloned into a pGEX
expression vector to create a vector encoding a fusion protein
comprising, from the N-terminus to the C-terminus, GST-thrombin cleavage
site-X protein. The fusion protein can be purified by affinity
chromatography using glutathione-agarose resin. Recombinant PKSRP unfused
to GST can be recovered by cleavage of the fusion protein with thrombin.

[0090]One strategy to maximize recombinant protein expression is to
express the protein in a host bacteria with an impaired capacity to
proteolytically cleave the recombinant protein (Gottesman, S., Gene
Expression Technology: Methods in Enzymology 185, Academic Press, San
Diego, Calif. (1990) 119-128). Another strategy is to alter the sequence
of the nucleic acid to be inserted into an expression vector so that the
individual codons for each amino acid are those preferentially utilized
in the bacterium chosen for expression, such as C. glutamicum (Wada et
al., 1992 Nucleic Acids Res. 20:2111-2118). Such alteration of nucleic
acid sequences of the invention can be carried out by standard DNA
synthesis techniques.

[0092]Alternatively, the PKSRPs of the invention can be expressed in
insect cells using baculovirus expression vectors. Baculovirus vectors
available for expression of proteins in cultured insect cells (e.g., Sf 9
cells) include the pAc series (Smith et al., 1983 Mol. Cell. Biol.
3:2156-2165) and the pVL series (Lucklow and Summers, 1989 Virology
170:31-39).

[0096]A plant expression cassette preferably contains regulatory sequences
capable of driving gene expression in plant cells and operably linked so
that each sequence can fulfill its function, for example, termination of
transcription by polyadenylation signals. Preferred polyadenylation
signals are those originating from Agrobacterium tumefaciens t-DNA such
as the gene 3 known as octopine synthase of the Ti-plasmid pTiACH5
(Gielen et al., 1984 EMBO J. 3:835) or functional equivalents thereof but
also all other terminators functionally active in plants are suitable.

[0097]As plant gene expression is very often not limited on
transcriptional levels, a plant expression cassette preferably contains
other operably linked sequences like translational enhancers such as the
overdrive-sequence containing the 5'-untranslated leader sequence from
tobacco mosaic virus enhancing the protein per RNA ratio (Gallie et al.,
1987 Nucl. Acids Research 15:8693-8711).

[0098]Plant gene expression has to be operably linked to an appropriate
promoter conferring gene expression in a timely, cell or tissue specific
manner. Preferred are promoters driving constitutive expression (Benfey
et al, 1989 EMBO J. 8:2195-2202) like those derived from plant viruses
like the 35S CAMV (Franck et al., 1980 Cell 21:285-294), the 19 S CaMV
(see also U.S. Pat. No. 5,352,605 and PCT Application No. WO 8402913) or
plant promoters like those from Rubisco small subunit described in U.S.
Pat. No. 4,962,028.

[0099]Other preferred sequences for use in plant gene expression cassettes
are targeting-sequences necessary to direct the gene product in its
appropriate cell compartment (for review see Kermode, 1996 Crit. Rev.
Plant Sci. 15(4):285-423 and references cited therein) such as the
vacuole, the nucleus, all types of plastids like amyloplasts,
chloroplasts, chromoplasts, the extracellular space, mitochondria, the
endoplasmic reticulum, oil bodies, peroxisomes and other compartments of
plant cells.

[0103]Also especially suited are promoters that confer plastid-specific
gene expression since plastids are the compartment where lipid
biosynthesis occurs. Suitable promoters are the viral RNA-polymerase
promoter described in PCT Application No. WO 95/16783 and PCT Application
No. WO 97/06250 and the clpP-promoter from Arabidopsis described in PCT
Application No. WO 99/46394.

[0104]The invention further provides a recombinant expression vector
comprising a PKSRP DNA molecule of the invention cloned into the
expression vector in an antisense orientation. That is, the DNA molecule
is operatively linked to a regulatory sequence in a manner that allows
for expression (by transcription of the DNA molecule) of an RNA molecule
that is antisense to a PKSRP mRNA. Regulatory sequences operatively
linked to a nucleic acid molecule cloned in the antisense orientation can
be chosen which direct the continuous expression of the antisense RNA
molecule in a variety of cell types. For instance, viral promoters and/or
enhancers, or regulatory sequences can be chosen which direct
constitutive, tissue specific or cell type specific expression of
antisense RNA. The antisense expression vector can be in the form of a
recombinant plasmid, phagemid or attenuated virus wherein antisense
nucleic acids are produced under the control of a high efficiency
regulatory region. The activity of the regulatory region can be
determined by the cell type into which the vector is introduced. For a
discussion of the regulation of gene expression using antisense genes see
Weintraub, H. et al., Antisense RNA as a molecular tool for genetic
analysis, Reviews--Trends in Genetics, Vol. 1(1)1986 and Mol et al., 1990
FEBS Letters 268:427-430.

[0105]Another aspect of the invention pertains to host cells into which a
recombinant expression vector of the invention has been introduced. The
terms "host cell" and "recombinant host cell" are used interchangeably
herein. It is understood that such terms refer not only to the particular
subject cell but they also apply to the progeny or potential progeny of
such a cell. Because certain modifications may occur in succeeding
generations due to either mutation or environmental influences, such
progeny may not, in fact, be identical to the parent cell, but are still
included within the scope of the term as used herein.

[0106]A host cell can be any prokaryotic or eukaryotic cell, For example,
a PKSRP can be expressed in bacterial cells such as C. glutamicum, insect
cells, fungal cells or mammalian cells (such as Chinese hamster ovary
cells (CHO) or COS cells), algae, ciliates, plant cells, fungi or other
microorganisms like C. glutamicum. Other suitable host cells are known to
those skilled, in the art.

[0108]In particular, the invention provides a method of producing a
transgenic plant with a PKSRP coding nucleic acid, wherein expression of
the nucleic acid(s) in the plant results in increased tolerance to
environmental stress as compared to a wild type variety of the plant
comprising: (a) transforming a plant cell with an expression vector
comprising a PKSRP nucleic acid, and (b) generating from the plant cell a
transgenic plant with a increased tolerance to environmental stress as
compared to a wild type variety of the plant. The invention also provides
a method of increasing expression of a gene of interest within a host
cell as compared to a wild type variety of the host cell, wherein the
gene of interest is transcribed in response to a PKSRP, comprising: (a)
transforming the host cell with an expression vector comprising a PKSRP
coding nucleic acid, and (b) expressing the PKSRP within the host cell,
thereby increasing the expression of the gene transcribed in response to
the PKSRP, as compared to a wild type variety of the host cell.

[0109]For such plant transformation, binary vectors such as pBinAR can be
used (Hofgen and Willmitzer, 1990 Plant Science 66:221-230). Construction
of the binary vectors can be performed by ligation of the cDNA in sense
or antisense orientation into the T-DNA. 5-prime to the cDNA a plant
promoter activates transcription of the cDNA. A polyadenylation sequence
is located 3-prime to the cDNA. Tissue-specific expression can be
achieved by using a tissue specific promoter. For example, seed-specific
expression can be achieved by cloning the napin or LeB4 or USP promoter
5-prime to the cDNA. Also, any other seed specific promoter element can
be used. For constitutive expression within the whole plant, the CaMV 35S
promoter can be used. The expressed protein can be targeted to a cellular
compartment using a signal peptide, for example for plastids,
mitochondria or endoplasmic reticulum (Kermode, 1996 Crit. Rev. Plant
Sci. 4(15):285-423). The signal peptide is cloned 5-prime in frame to the
cDNA to archive subcellular localization of the fusion protein.
Additionally, promoters that are responsive to abiotic stresses can be
used with, such as the Arabidopsis promoter RD29 A, the nucleic acid
sequences disclosed herein. One skilled in the art will recognize that
the promoter used should be operatively linked to the nucleic acid such
that the promoter causes transcription of the nucleic acid which results
in the synthesis of an mRNA which encodes a polypeptide. Alternatively,
the RNA can be an antisense RNA for use in affecting subsequent
expression of the same or another gene or genes.

[0110]Alternate methods of transfection include the direct transfer of DNA
into developing flowers via electroporation or Agrobacterium mediated
gene transfer. Agrobacterium mediated plant transformation can be
performed using for example the GV3101 (pMP90) (Koncz and Schell, 1986
Mol. Gen. Genet. 204:383-396) or LBA4404 (Clontech) Agrobacterium
tumefaciens strain. Transformation can be performed by standard
transformation and regeneration techniques (Deblaere et al., 1994 Nucl.
Acids. Res. 13:4777-4788; Gelvin, Stanton B. and Schilperoort, Robert A,
Plant Molecular Biology Manual, 2nd Ed.--Dordrecht: Kluwer Academic
Publ., 1995.--in Sect., Ringbuc Zentrale Signatur: BT11-P ISBN
0-7923-2731-4; Glick, Bernard R.; Thompson, John E., Methods in Plant
Molecular Biology and Biotechnology, Boca Raton: CRC Press, 1993.--360
S., ISBN 0-8493-5164-2).; For example, rapeseed can be transformed via
cotyledon or hypocotyl transformation (Moloney et al., 1989 Plant cell
Report 8:238-242; De Block et al., 1989 Plant Physiol. 91:694-701). Use
of antibiotica for Agrobacterium and plant selection depends on the
binary vector and the Agrobacterium strain used for transformation.
Rapeseed selection is normally performed using kanamycin as selectable
plant marker. Agrobacterium mediated gene transfer to flax can be
performed using, for example, a technique described by Mlynarova et al.,
1994 Plant Cell Report 13:282-285. Additionally, transformation of
soybean can be performed using for example a technique described in
European Patent No. 0424 047, U.S. Pat. No. 5,322,783, European Patent
No. 0397 687, U.S. Pat. No. 5,376,543 or U.S. Pat. No. 5,169,770.
Transformation of maize can be achieved by particle bombardment,
polyethylene glycol mediated DNA uptake or via the silicon carbide fiber
technique. (See, for example, Freeling and Walbot "The maize handbook"
Springer Verlag: New York (1993) ISBN 3-540-97826-7). A specific example
of maize transformation is found in U.S. Pat. No. 5,990,387 and a
specific example of wheat transformation can be found in PCT Application
No. WO 93/07256.

[0111]For stable transfection of mammalian cells, it is known that,
depending upon the expression vector and transfection technique used,
only a small fraction of cells may integrate the foreign DNA into their
genome. In order to identify and select these integrants, a gene that
encodes a selectable marker (e.g., resistance to antibiotics) is
generally introduced into the host cells along with the gene of interest.
Preferred selectable markers include those which confer resistance to
drugs, such as G418, hygromycin and methotrexate or in plants that confer
resistance towards a herbicide such as glyphosate or glufosinate. Nucleic
acid molecules encoding a selectable marker can be introduced into a host
cell on the same vector as that encoding a PKSRP or can be introduced on
a separate vector. Cells stably transfected with the introduced nucleic
acid molecule can be identified by, for example, drug selection (e.g.,
cells that have incorporated the selectable marker gene will survive,
while the other cells die).

[0112]To create a homologous recombinant microorganism, a vector is
prepared which contains at least a portion of a PKSRP gene into which a
deletion, addition or substitution has been introduced to thereby alter,
e.g., functionally disrupt, the PKSRP gene. Preferably, the PKSRP gene is
a Physcomitrella patens PKSRP gene, but it can be a homolog from a
related plant or even from a mammalian, yeast, or insect source. In a
preferred embodiment, the vector is designed such that, upon homologous
recombination, the endogenous PKSRP gene is functionally disrupted (i.e.,
no longer encodes a functional protein; also referred to as a knock-out
vector). Alternatively, the vector can be designed such that, upon
homologous recombination, the endogenous PKSRP gene is mutated or
otherwise altered but still encodes a functional protein (e.g., the
upstream regulatory region can be altered to thereby alter the expression
of the endogenous PKSRP). To create a point mutation via homologous
recombination, DNA-RNA hybrids can be used in a technique known as
chimeraplasty (Cole-Strauss et al., 1999 Nucleic Acids Research 27(5):
1323-1330 and Kmiec, 1999 Gene therapy American Scientist.
87(3):240-247). Homologous recombination procedures in Physcomitrella
patens are also well known in the art and are contemplated for use
herein.

[0113]Whereas in the homologous recombination vector, the altered portion
of the PKSRP gene is flanked at its 5' and 3' ends by an additional
nucleic acid molecule of the PKSRP gene to allow for homologous
recombination to occur between the exogenous PKSRP gene carried by the
vector and an endogenous PKSRP gene, in a microorganism or plant. The
additional flanking PKSRP nucleic acid molecule is of sufficient length
for successful homologous recombination with the endogenous gene.
Typically, several hundreds of base pairs up to kilobases of flanking DNA
(both at the 5' and 3' ends) are included in the vector (see e.g.,
Thomas, K. R., and Capecchi, M. R., 1987 Cell 51:503 for a description of
homologous recombination vectors or Strepp et al., 1998 PNAS, 95
(8):4368-4373 for cDNA based recombination in Physcomitrella patens). The
vector is introduced into a microorganism or plant cell (e.g., via
polyethylene glycol mediated DNA), and cells in which the introduced
PKSRP gene has homologously recombined with the endogenous PKSRP gene are
selected using art-known techniques.

[0114]In another embodiment, recombinant microorganisms can be produced
that contain selected systems which allow for regulated expression of the
introduced gene. For example, inclusion of a PKSRP gene on a vector
placing it under control of the lac operon permits expression of the
PKSRP gene only in the presence of IPTG, Such regulatory systems are well
known in the art.

[0115]A host cell of the invention, such as a prokaryotic or eukaryotic
host cell in culture, can be used to produce (i.e., express) a PKSRP.
Accordingly, the invention further provides methods for producing PKSRPs
using the host cells of the invention. In one embodiment, the method
comprises culturing the host cell of invention (into which a recombinant
expression vector encoding a PKSRP has been introduced, or into which
genome has been introduced a gene encoding a wild-type or altered PKSRP )
in a suitable medium until PKSRP is produced. In another embodiment, the
method further comprises isolating PKSRPs from the medium or the host
cell.

[0116]Another aspect of the invention pertains to isolated PKSRPs, and
biologically active portions thereof. An "isolated" or "purified" protein
or biologically active portion thereof is free of some of the cellular
material when produced by recombinant DNA techniques, or chemical
precursors or other chemicals when chemically synthesized. The language
"substantially free of cellular material" includes preparations of PKSRP
in which the protein is separated from some of the cellular components of
the cells in which it is naturally or recombinantly produced. In one
embodiment, the language "substantially free of cellular material"
includes preparations of a PKSRP having less than about 30% (by dry
weight) of non-PKSRP material (also referred to herein as a
"contaminating protein"), more preferably less than about 20% of
non-PKSRP material, still more preferably less than about 10% of
non-PKSRP material, and most preferably less than about 5% non-PKSRP
material.

[0117]When the PKSRP or biologically active portion thereof is
recombinantly produced, it is also preferably substantially free of
culture medium, i.e., culture medium represents less than about 20%, more
preferably less than about 10%, and most preferably less than about 5% of
the volume of the protein preparation. The language "substantially free
of chemical precursors or other chemicals" includes preparations of PKSRP
in which the protein is separated from chemical precursors or other
chemicals that are involved in the synthesis of the protein. In one
embodiment, the language "substantially free of chemical precursors or
other chemicals" includes preparations of a PKSRP having less than about
30% (by dry weight) of chemical precursors or non-PKSRP chemicals, more
preferably less than about 20% chemical precursors or non-PKSRP
chemicals, still more preferably less than about 10% chemical precursors
or non-PKSRP chemicals, and most preferably less than about 5% chemical
precursors or non-PKSRP chemicals. In preferred embodiments, isolated
proteins, or biologically active portions thereof, lack contaminating
proteins from the same organism from which the PKSRP is derived.
Typically, such proteins are produced by recombinant expression of, for
example, a Physcomitrella patens PKSRP in plants other than
Physcomitrella patens or microorganisms such as C. glutamicum, ciliates,
algae or fungi.

[0118]The nucleic acid molecules, proteins, protein homologs, fusion
proteins, primers, vectors, and host cells described herein can be used
in one or more of the following methods: identification of Physcomitrella
patens and related organisms; mapping of genomes of organisms related to
Physcomitrella patens; identification and localization of Physcomitrella
patens sequences of interest; evolutionary studies; determination of
PKSRP regions required for function; modulation of a PKSRP activity;
modulation of the metabolism of one or more cell functions; modulation of
the transmembrane transport of one or more compounds; and modulation of
stress resistance.

[0119]The moss Physcomitrella patens represents one member of the mosses.
It is related to other mosses such as Ceratodon purpureus which is
capable of growth in the absence of light. Mosses like Ceratodon and
Physcomitrella share a high degree of homology on the DNA sequence and
polypeptide level allowing the use of heterologous screening of DNA
molecules with probes evolving from other mosses or organisms, thus
enabling the derivation of a consensus sequence suitable for heterologous
screening or functional annotation and prediction of gene functions in
third species. The ability to identify such functions can therefore have
significant relevance, e.g., prediction of substrate specificity of
enzymes. Further, these nucleic acid molecules may serve as reference
points for the mapping of moss genomes, or of genomes of related
organisms.

[0120]The PKSRP nucleic acid molecules of the invention have a variety of
uses. Most importantly, the nucleic acid and amino acid sequences of the
present invention can be used to transform plants, thereby inducing
tolerance to stresses such as drought, high salinity and cold. The
present invention therefore provides a transgenic plant transformed by a
PKSRP nucleic acid (coding or antisense), wherein expression of the
nucleic acid sequence in the plant results in increased tolerance to
environmental stress as compared to a wild type variety of the plant. The
transgenic plant can be a monocot or a dicot. The invention further
provides that the transgenic plant can be selected from maize, wheat,
rye, oat, triticale, rice, barley, soybean, peanut, cotton, rapeseed,
canola, manihot, pepper, sunflower, tagetes, solanaceous plants, potato,
tobacco, eggplant, tomato, Vicia species, pea, alfalfa, coffee, cacao,
tea, Salix species, oil palm, coconut, perennial grass and forage crops,
for example.

[0122]The present invention also provides methods of modifying stress
tolerance of a plant comprising, modifying the expression of a PKSRP in
the plant. The invention provides that this method can be performed such
that the stress tolerance is either increased or decreased. In
particular, the present invention provides methods of producing a
transgenic plant having an increased tolerance to environmental stress as
compared to a wild type variety of the plant comprising increasing
expression of a PKSRP in a plant.

[0123]The methods of increasing expression of PKSRPs can be used wherein
the plant is either transgenic or not transgenic. In cases when the plant
is transgenic, the plant can be transformed with a vector containing any
of the above described PKSRP coding nucleic acids, or the plant can be
transformed with a promoter that directs expression of native PKSRP in
the plant, for example. The invention provides that such a promoter can
be tissue specific. Furthermore, such a promoter can be developmentally
regulated. Alternatively, non-transgenic plants can have native PKSRP
expression modified by inducing a native promoter.

[0125]In addition to introducing the PKSRP nucleic acid sequences into
transgenic plants, these sequences can also be used to identify an
organism as being Physcomitrella patens or a close relative thereof.
Also, they may be used to identify the presence of Physcomitrella patens
or a relative thereof in a mixed population of microorganisms. The
invention provides the nucleic acid sequences of a number of
Physcomitrella patens genes; by probing the extracted genomic DNA of a
culture of a unique or mixed population of microorganisms under stringent
conditions with a probe spanning a region of a Physcomitrella patens gene
which is unique to this organism, one can ascertain whether this organism
is present.

[0126]Further, the nucleic acid and protein molecules of the invention may
serve as markers for specific regions of the genome. This has utility not
only in the mapping of the genome, but also in functional studies of
Physcomitrella patens proteins. For example, to identify the region of
the genome to which a particular Physcomitrella patens DNA-binding
protein binds, the Physcomitrella patens genome could be digested, and
the fragments incubated with the DNA-binding protein. Those fragments
that bind the protein may be additionally probed with the nucleic acid
molecules of the invention, preferably with readily detectable labels.
Binding of such a nucleic acid molecule to the genome fragment enables
the localization of the fragment to the genome map of Physcomitrella
patens, and, when performed multiple times with different enzymes,
facilitates a rapid determination of the nucleic acid sequence to which
the protein binds. Further, the nucleic acid molecules of the invention
may be sufficiently homologous to the sequences of related species such
that these nucleic acid molecules may serve as markers for the
construction of a genomic map in related mosses.

[0127]The PKSRP nucleic acid molecules of the invention are also useful
for evolutionary and protein structural studies. The metabolic and
transport processes in which the molecules of the invention participate
are utilized by a wide variety of prokaryotic and eukaryotic cells; by
comparing the sequences of the nucleic acid molecules of the present
invention to those encoding similar enzymes from other organisms, the
evolutionary relatedness of the organisms can be assessed, Similarly,
such a comparison permits an assessment of which regions of the sequence
are conserved and which are not, which may aid in determining those
regions of the protein that are essential for the functioning of the
enzyme. This type of determination is of value for protein engineering
studies and may give an indication of what the protein can tolerate in
terms of mutagenesis without losing function.

[0128]Manipulation of the PKSRP nucleic acid molecules of the invention
may result in the production of PKSRPs having functional differences from
the wild-type PKSRPs. These proteins may be improved in efficiency or
activity, may be present in greater numbers in the cell than is usual, or
may be decreased in efficiency or activity.

[0129]There are a number of mechanisms by which the alteration of a PKSRP
of the invention may directly affect stress response and/or stress
tolerance. In the case of plants expressing PKSRPs, increased transport
can lead to improved salt and/or solute partitioning within the plant
tissue and organs. By either increasing the number or the activity of
transporter molecules which export ionic molecules from the cell, it may
be possible to affect the salt tolerance of the cell.

[0131]For example, yeast expression vectors comprising the nucleic acids
disclosed herein, or fragments thereof, can be constructed and
transformed into Saccharomyces cerevisiae using standard protocols. The
resulting transgenic cells can then be assayed for fail or alteration of
their tolerance to drought, salt, and temperature stress. Similarly,
plant expression vectors comprising the nucleic acids disclosed herein,
or fragments thereof, can be constructed and transformed into an
appropriate plant cell such as Arabidopsis, soy, rape, maize, wheat,
Medicago truncatula, etc., using standard protocols. The resulting
transgenic cells and/or plants derived there from can then be assayed for
fail or alteration of their tolerance to drought, salt, and temperature
stress.

[0132]The engineering of one or more PKSRP genes of the invention may also
result in PKSRPs having altered activities which indirectly impact the
stress response and/or stress tolerance of algae, plants, ciliates or
fungi or other microorganisms like C. glutamicum. For example, the normal
biochemical processes of metabolism result in the production of a variety
of products (e.g., hydrogen peroxide and other reactive oxygen species)
which may actively interfere with these same metabolic processes (for
example, peroxynitrite is known to nitrate tyrosine side chains, thereby
inactivating some enzymes having tyrosine in the active site (Groves, J.
T., 1999 Curr. Opin. Chem. Biol. 3(2): 226-235). While these products are
typically excreted, cells can be genetically altered to transport more
products than is typical for a wild-type cell. By optimizing the activity
of one or more PKSRPs of the invention which are involved in the export
of specific molecules, such as salt molecules, it may be possible to
improve the stress tolerance of the cell.

[0133]Additionally, the sequences disclosed herein, or fragments thereof,
can be used to generate knockout mutations in the genomes of various
organisms, such as bacteria, mammalian cells, yeast cells, and plant
cells (Girke, T., 1998 The Plant Journal 15:39-48). The resultant
knockout cells can then be evaluated for their ability or capacity to
tolerate various stress conditions, their response to various stress
conditions, and the effect on the phenotype and/or genotype of the
mutation. For other methods of gene inactivation see U.S. Pat. No.
6,004,804 "Non-Chimeric Mutational Vectors" and Puttaraju et al., 1999
Spliceosome-mediated RNA trans-splicing as a tool for gene therapy Nature
Biotechnology 17:246-252.

[0134]The aforementioned mutagenesis strategies for PKSRPs resulting in
increased stress resistance are not meant to be limiting; variations on
these strategies will be readily apparent to one skilled in the art.
Using such strategies, and incorporating the mechanisms disclosed herein,
the nucleic acid and protein molecules of the invention may be utilized
to generate algae, ciliates, plants, fungi or other microorganisms like
C. glutamicum expressing mutated PKSRP nucleic acid and protein molecules
such that the stress tolerance is improved.

[0135]The present invention also provides antibodies that specifically
bind to a PKSRP, or a portion thereof, as encoded by a nucleic acid
described herein. Antibodies can be made by many well-known methods (See,
e.g. Harlow and Lane, "Antibodies; A Laboratory Manual" Cold Spring
Harbor Laboratory, Cold Spring Harbor, N.Y., (1988)). Briefly, purified
antigen can be injected into an animal in an amount and in intervals
sufficient to elicit an immune response. Antibodies can either be
purified directly, or spleen cells can be obtained from the animal. The
cells can then fused with an immortal cell line and screened for antibody
secretion. The antibodies can be used to screen nucleic acid clone
libraries for cells secreting the antigen. Those positive clones can then
be sequenced. (See, for example, Kelly et al., 1992 Bio/Technology
10:163-167; Bebbington et al., 1992 Bio/Technology 10:169-175).

[0136]The phrases "selectively binds" and "specifically binds" with the
polypeptide refer to a binding reaction that is determinative of the
presence of the protein in a heterogeneous population of proteins and
other biologies. Thus, under designated immunoassay conditions, the
specified antibodies bound to a particular protein do not bind in a
significant amount to other proteins present in the sample. Selective
binding of an antibody under such conditions may require an antibody that
is selected for its specificity for a particular protein. A variety of
immunoassay formats may be used to select antibodies that selectively
bind with a particular protein. For example, solid-phase ELISA
immunoassays are routinely used to select antibodies selectively
immunoreactive with a protein. See Harlow and Lam "Antibodies, A
Laboratory Manual" Cold Spring Harbor Publications, New York, (1988), for
a description of immunoassay formats and conditions that could be used to
determine selective binding.

[0137]In some instances, it is desirable to prepare monoclonal antibodies
from various hosts. A description of techniques for preparing such
monoclonal antibodies may be found in Stites et al., editors, "Basic and
Clinical Immunology," (Lange Medical Publications, Los Altos, Calif.,
Fourth Edition) and references cited therein, and in Harlow and Lane
("Antibodies, A Laboratory Manual" Cold Spring Harbor Publications, New
York, 1988).

[0138]Throughout this application, various publications are referenced.
The disclosures of all of these publications and those references cited
within those publications in their entireties are hereby incorporated by
reference into this application in order to more fully describe the state
of the art to which this invention pertains.

[0139]It should also be understood that the foregoing relates to preferred
embodiments of the present invention and that numerous changes may be
made therein without departing from the scope of the invention. The
invention is further illustrated by the following examples, which are not
to be construed in any way as imposing limitations upon the scope
thereof. On the contrary, it is to be clearly understood that resort may
be had to various other embodiments, modifications, and equivalents
thereof, which, after reading the description herein, may suggest
themselves to those skilled in the art without departing from the spirit
of the present invention and/or the scope of the appended claims.

EXAMPLES

Example 1

Growth of Physcomitrella patens Cultures

[0140]For this study, plants of the species Physcomitrella patens (Hedw.)
B. S. G. from the collection of the genetic studies section of the
University of Hamburg were used. They originate from the strain 16/14
collected by H. L. K. Whitehouse in Gransden Wood, Huntingdonshire
(England), which was subcultured from a spore by Engel (1968, Am. J. Bot.
55, 438-446). Proliferation of the plants was carried out by means of
spores and by means of regeneration of the gametophytes. The protonema
developed from the haploid spore as a chloroplast-rich chloronema and
chloroplast-low caulonema, on which buds formed after approximately 12
days. These grew to give gametophores bearing antheridia and archegonia.
After fertilization, the diploid sporophyte with a short seta and the
spore capsule resulted, in which the meiospores matured.

[0141]Culturing was carried out in a climatic chamber at an air
temperature of 25° C. and light intensity of 55 micromol s-1
m-2 (white light; Philips TL 65W/25 fluorescent tube) and a
light/dark change of 16/8 hours. The moss was either modified in liquid
culture using Knop medium according to Reski and Abel (1985, Planta
165:354-358) or cultured on Knop solid medium using 1% oxoid agar
(Unipath, Basingstoke, England). The protonemas used for RNA and DNA
isolation were cultured in aerated liquid cultures, The protonemas were
comminuted every 9 days and transferred to fresh culture medium.

[0143]The plant material was triturated under liquid nitrogen in a mortar
to give a fine powder and transferred to 2 ml Eppendorf vessels, The
frozen plant material was then covered with a layer of 1 ml of
decomposition buffer (1 ml CTAB buffer, 100 μl of N-laurylsarcosine
buffer, 20 μl of β-mercaptoethanol and 10 μl of proteinase K
solution, 10 mg/ml) and incubated at 60° C. for one hour with
continuous shaking. The homogenate obtained was distributed into two
Eppendorf vessels (2 ml) and extracted twice by shaking with the same
volume of chloroform/isoamyl alcohol (24:1). For phase separation,
centrifugation was carried out at 8000×g and room temperature for
15 minutes in each case. The DNA was then precipitated at -70° C.
for 30 minutes using ice-cold isopropanol. The precipitated DNA was
sedimented at 4° C. and 10,000 g for 30 minutes and resuspended in
180 μl of TE buffer (Sambrook et al., 1989, Cold Spring Harbor
Laboratory Press: ISBN 0-87969-309-6). For further purification, the DNA
was treated with NaCl (1.2 M final concentration) and precipitated again
at -70° C. for 30 minutes using twice the volume of absolute
ethanol. After a washing step with 70% ethanol, the DNA was dried and
subsequently taken up in 50 μl of H2 O+RNAse (50 mg/ml final
concentration). The DNA was dissolved overnight at 4° C. and the
RNAse digestion was subsequently carried out at 37° C. for 1 hour.
Storage of the DNA took place at 4° C.

Example 3

β Isolation of Total RNA and Poly-(A)+ RNA and cDNA Library
Construction from Physcomitrella patens

[0144]For the investigation of transcripts, both total RNA and poly-(A)+
RNA were isolated. The total RNA was obtained from wild-type 9 day old
protonemata following the GTC-method (Reski et al. 1994, Mol. Gen.
Genet., 244:352-359). The Poly(A)+ RNA was isolated using Dyna
BeadsR (Dynal, Oslo, Norway) following the instructions of the
manufacturers protocol. After determination of the concentration of the
RNA or of the poly(A)+ RNA, the RNA was precipitated by addition of 1/10
volumes of 3 M sodium acetate pH 4.6 and 2 volumes of ethanol and stored
at -70° C.

[0153]The plasmid construct pACGH101 was digested with PstI (Roche) and
FseI (NEB) according to manufacturers' instructions. The fragment was
purified by agarose gel and extracted via the Qiaex II DNA Extraction kit
(Qiagen). This resulted in a vector fragment with the Arabidopsis Actin2
promoter with internal intron and the OCS3 terminator. Primers for PCR
amplification of the NPTII gene were designed as follows:

[0155]The NPT-Topo construct was then digested with PstI (Roche) and FseI
(NEB) according to manufacturers' instructions. The 0.9 kilobase fragment
was purified on agarose gel and extracted by Qiaex II DNA Extraction kit
(Qiagen). The Pst/Fse insert fragment from NPT-Topo and the Pst/Fse
vector fragment from pACGH101 were then ligated together using T4 DNA
Ligase (Roche) following manufacturer's instructions. The ligation was
then transformed into Top10 cells (Invitrogen) under standard conditions,
creating pBPSsc019 construct. Colonies were selected on LB plates with 50
ug/ml kanamycin sulfate and grown overnight at 37° C., These
colonies were then used to inoculate 2 ml LB media with 50 ug/ml
kanamycin sulfate and grown overnight at 37° C., Plasmid DNA was
recovered using the Qiaprep Spin Miniprep kit (Qiagen) following the
manufacturer's instructions.

[0156]The pBPSSC019 construct was digested with KpnI and BsaI (Roche)
according to manufacturer's instructions. The fragment was purified via
agarose gel and then extracted via the Qiaex II DNA Extraction kit
(Qiagen) as per its instructions, resulting in a 3 kilobase Act-NPT
cassette, which included the Arabidopsis Actin2 promoter with internal
intron, the NPTII gene and the OCS3 terminator.

[0157]The pBPSJH001 vector was digested with SpeI and ApaI (Roche) and
blunt-end filled with Klenow enzyme and 0.1 mM dNTPs (Roche) according to
manufacture's instructions. This produced a 10.1 kilobase vector fragment
minus the Gentamycin cassette, which was recircularized by self-ligating
with T4 DNA Ligase (Roche), and transformed into Top10 cells (Invitrogen)
via standard conditions. Transformed cells were selected for on LB agar
containing 50 μg/ml kanamycin sulfate and grown overnight at
37° C., Colonies were then used to inoculate 2 ml of liquid LB
containing 50 μg/ml kanamycin sulfate and grown overnight at
37° C. Plasmid DNA was extracted using the QIAprep Spin Miniprep
Kit (Qiagen) following manufacture's instructions. The recircularized
plasmid was then digested with KpnI (Roche) and extracted from agarose
gel via the Qiaex II DNA Extraction kit (Qiagen) as per manufacturer's
instructions.

[0158]The Act-NPT Kpn-cut insert and the Kpn-cut pBPSJH001 recircularized
vector were then ligated together using T4 DNA Ligase (Roche) and
transformed into Top10 cells (Invitrogen) as per manufacturers'
instructions. The resulting construct, pBPSsc022, now contained the Super
Promoter, the GUS gene, the NOS terminator, and the Act-NPT cassette.
Transformed cells were selected for on LB agar containing 50 μg/ml
kanmycin sulfate and grown overnight at 37° C. Colonies were then
used to inoculate 2 ml of liquid LB containing 50 μg/ml kanamycin
sulfate and grown overnight at 37° C. Plasmid DNA was extracted
using the QIAprep Spin Miniprep Kit (Qiagen) following manufacturer's
instructions. After confirmation of ligation success via restriction
digests, pBPSsc022 plasmid DNA was further propigated and recovered using
the Plasmid Midiprep Kit (Qiagen) following the manufacturer's
instructions.

[0159]The fragments containing the different Physcomitrella patens protein
kinases were subcloned from the recombinant PCR2.1 TOPO vectors by double
digestion with restriction enzymes (see Table 16) according to
manufacturer's instructions. The subsequence fragment was excised from
agarose gel with a QIAquick Gel Extraction Kit (QIAgen) according to
manufacture's instructions and ligated into the binary vectors pGMSG,
cleaved with XmaI and Ecl136II and dephosphorylated prior to ligation.
The resulting recombinant pGMSG contained the corresponding transcription
factor in the sense orientation under the constitutive super promoter,

[0163]T1 seedlings were transferred to dry, sterile filter paper in a
petri dish and allowed to desiccate for two hours at 80% RH (relative
humidity) in a Percieval Growth Cabinet MLR-350H, micromole s-1
m-2 (white light; Philips TL 65W/25 fluorescent tube). The RH was
then decreased to 60% and the seedlings were desiccated further for eight
hours. Seedlings were then removed and placed on 1/2 MS 0.6% agar plates
supplemented with 2 μg/ml benomyl (Sigma-Aldrich) and 0.5 g/L MES
(Sigma-Aldrich) and scored after five days.

[0165]Seedlings were moved to petri dishes containing 1/2 MS 0.6% agar
supplemented with 2% sucrose and 2 μg/ml benomyl. After four days, the
seedlings were incubated at 4° C. for 1 hour and then covered with
shaved ice. The seedlings were then placed in an Environmental Specialist
ES2000 Environmental Chamber and incubated for 3.5 hours beginning at
-1.0° C. decreasing 1° C./hour. The seedlings were then
incubated at -5.0° C. for 24 hours and then allowed to thaw at
5° C. for 12 hours. The water was poured off and the seedlings
were scored after 5 days.

[0167]Seedlings were transferred to filter paper soaked in 1/2 MS and
placed on 1/2 MS 0.6% agar supplemented with 2 μg/ml benomyl the night
before the salt tolerance screening. For the salt tolerance screening,
the filter paper with the seedlings was moved to stacks of sterile filter
paper, soaked in 50 mM NaCl, in a petri dish. After two hours, the filter
paper with the seedlings was moved to stacks of sterile filter paper,
soaked with 200 mM NaCl, in a petri dish. After two hours, the filter
paper with the seedlings was moved to stacks of sterile filter paper,
soaked in 600 mM NaCl, in a petri dish. After 10 hours, the seedlings
were moved to petri dishes containing 1/2 MS 0.6% agar supplemented with
2 μg/ml benomyl. The seedlings were scored after 5 days.

[0169]One leaf from a wild type and a transgenic Arabidopsis plant was
homogenized in 250 μl Hexadecyltrimethyl ammonium bromide (CTAB)
buffer (2% CTAB, 1.4 M NaCl, 8 mM EDTA and 20 mM Tris pH 8.0) and 1 μl
β-mercaptoethanol. The samples were incubated at 60-65° C.
for 30 minutes and 250 μl of Chloroform was then added to each sample.
The samples were vortexed for 3 minutes and centrifuged for 5 minutes at
18,000×g. The supernatant was taken from each sample and 150 μl
isopropanol was added. The samples were incubated at room temperature for
15 minutes, and centrifuged for 10 minutes at 18,000×g. Each pellet
was washed with 70% ethanol, dried, and resuspended in 20 μl TE. 4
μl of above suspension was used in a 20 μl PCR reaction using Taq
DNA polymerase (Roche Molecular Biochemicals) according to the
manufacturer's instructions.

[0170]Binary vector plasmid with each gene cloned in was used as positive
control, and the wild-type C24 genomic DNA was used as negative control
in the PCR reactions. 10 μl PCR reaction was analyzed on 0.8%
agarose-ethidium bromide gel.

[0172]The PCR program was as following: 30 cycles of 1 minute at
94° C., 1 minute at 62° C. and 4 minutes at 72° C.,
followed by 10 minutes at 72° C. A 2.8 kb fragment was produced
from the positive control and the transgenic plants.

[0174]The primers were used in the first round of reactions with the
following program: 30 cycles of 1 minute at 94° C., 1 minute at
62° C. and 4 minutes at 72° C., followed by 10 minutes at
72° C. A 1.1 kb fragment was generated from the positive control
and the T1 transgenic plants.

[0176]The PCR program was as following: 30 cycles of 1 minute at
94° C., 1 minute at 62° C. and 4 minutes at 72° C.,
followed by 10 minutes at 72° C. A 1.6 kb fragment was produced
from the positive control and the transgenic plants.

[0178]The PCR program was as following: 30 cycles of 1 minute at
94° C., 1 minute at 62° C. and 4 minutes at 72° C.,
followed by 10 minutes at 72° C. A 1.4 kb fragment was produced
from the positive control and the transgenic plants.

[0180]The PCR program was as following: 30 cycles of 1 minute at
94° C., 1 minute at 62° C. and 4 minutes at 72° C.,
followed by 10 minutes at 72° C. A 1.7 kb fragment was produced
from the positive control and the transgenic plants.

[0182]The PCR program was as following: 30 cycles of 1 minute at
94° C., 1 minute at 62° C. and 4 minutes at 72° C.,
followed by 10 minutes at 72° C. A 1.9 kb fragment was produced
from the positive control and the transgenic plants.

[0184]The PCR program was as following: 30 cycles of 1 minute at
94° C., 1 minute at 62° C. and 4 minutes at 72° C.,
followed by 10 minutes at 72° C. A 1.2 kb fragment was produced
from the positive control and the transgenic plants.

[0186]The PCR program was as following: 30 cycles of 1 minute at
94° C., 1 minute at 62° C. and 4 minutes at 72° C.,
followed by 10 minutes at 72° C. A 1.7 kb fragment was produced
from the positive control and the transgenic plants.

[0188]The PCR program was as following: 30 cycles of 1 minute at
94° C., 1 minute at 62° C. and 4 minutes at 72° C.,
followed by 10 minutes at 72° C. A 2.2 kb fragment was produced
from the positive control and the transgenic plants.

[0190]The PCR program was as following: 30 cycles of 1 minute at
94° C., 1 minute at 62° C. and 4 minutes at 72° C.,
followed by 10 minutes at 72° C. A 1.7 kb fragment was produced
from the positive control and the transgenic plants.

[0192]The PCR program was as following: 30 cycles of 1 minute at
94° C., 1 minute at 62° C. and 4 minutes at 72° C.,
followed by 10 minutes at 72° C. A 1.4 kb fragment was produced
from the positive control and the transgenic plants,

[0194]The PCR program was as following: 30 cycles of 1 minute at
94° C., 1 minute at 62° C. and 4 minutes at 72° C.,
followed by 10 minutes at 72° C. A 2.3 kb fragment was produced
from the positive control and the transgenic plants,

[0196]The PCR program was as following: 30 cycles of 1 minute at
94° C., 1 minute at 62° C. and 4 minutes at 72° C.,
followed by 10 minutes at 72° C. A 2.2 kb fragment was produced
from the positive control and the transgenic plants.

[0197]The transgenes were successfully amplified from the T1 transgenic
lines, but not from the wild type C24. This result indicates that the T1
transgenic plants contain at least one copy of the transgenes. There was
no indication of existence of either identical or very similar genes in
the untransformed Arabidopsis thaliana control which could be amplified
by this method.

[0198]Transgene expression was detected using RT-PCR. Total RNA was
isolated from stress-treated plants using a procedure adapted from
(Verwoerd et al., 1989 NAR 17:2362). Leaf samples (50-100 mg) were
collected and ground to a fine powder in liquid nitrogen. Ground tissue
was resuspended in 500 μl of a 80° C., 1:1 mixture, of phenol
to extraction buffer (100 mM LiCl, 100 mM Tris pH8, 10 mM EDTA, 1% SDS),
followed by brief vortexing to mix. After the addition of 250 μl of
chloroform, each sample was vortexed briefly. Samples were then
centrifuged for 5 minutes at 12,000×g. The upper aqueous phase was
removed to a fresh eppendorf tube. RNA was precipitated by adding
1/10th volume 3M sodium acetate and 2 volumes 95% ethanol. Samples
were mixed by inversion and placed on ice for 30 minutes. RNA was
pelleted by centrifugation at 12,000×g for 10 minutes. The
supernatant was removed and pellets briefly air-dried. RNA sample pellets
were resuspended in 10 μl DEPC treated water. To remove contaminating
DNA from the samples, each was treated with RNase-free DNase (Roche)
according to the manufacturer's recommendations. cDNA was synthesized
from total RNA using the 1st Strand cDNA synthesis kit (Boehringer
Mannheim) following manufacturer's recommendations.

[0200]Expression of the transgenes was detected in the T1 transgenic line.
This result indicated that the transgenes are expressed in the transgenic
lines and strongly suggested that their gene product improved plant
stress tolerance in the transgenic line. On the other hand, no expression
of identical or very similar endogenous genes could be detected by this
method. These results are in agreement with the data from Example 7. This
greatly supports our statement that the observed stress tolerance is due
to the introduced transgene.

[0202]Expression of the transgenes was detected in the T1 transgenic line.
These results indicated that the transgenes are expressed in the
transgenic lines and strongly suggested that their gene product improved
plant stress tolerance in the transgenic lines. In agreement with the
previous statement, no expression of identical or very similar endogenous
genes could be detected by this method. These results are in agreement
with the data from Example 7.

[0204]Seeds of soybean are surface sterilized with 70% ethanol for 4
minutes at room temperature with continuous shaking, followed by 20%
(v/v) Clorox supplemented with 0.05% (v/v) Tween for 20 minutes with
continuous shaking. Then, the seeds are rinsed 4 times with distilled
water and placed on moistened sterile filter paper in a Petri dish at
room temperature for 6 to 39 hours. The seed coats are peeled off, and
cotyledons are detached from the embryo axis. The embryo axis is examined
to make sure that the meristematic region is not damaged. The excised
embryo axes are collected in a half-open sterile Petri dish and air-dried
to a moisture content less than 20% (fresh weight) in a sealed Petri dish
until further use.

[0205]Agrobacterium tumefaciens culture is prepared from a single colony
in LB solid medium plus appropriate antibiotics (e.g. 100 mg/l
streptomycin, 50 mg/l kanamycin) followed by growth of the single colony
in liquid LB medium to an optical density at 600 nm of 0.8. Then, the
bacteria culture is pelleted at 7000 rpm for 7 minutes at room
temperature, and resuspended in MS (Murashige and Skoog, 1962) medium
supplemented with 100 μM acetosyringone. Bacteria cultures are
incubated in this pre-induction medium for 2 hours at room temperature
before use. The axis of soybean zygotic seed embryos at approximately 15%
moisture content are imbibed for 2 hours at room temperature with the
pre-induced Agrobacterium suspension culture. The embryos are removed
from the imbibition culture and are transferred to Petri dishes
containing solid MS medium supplemented with 2% sucrose and incubated for
2 days, in the dark at room temperature. Alternatively, the embryos are
placed on top of moistened (liquid MS medium) sterile filter paper in a
Petri dish and incubated under the same conditions described above. After
this period, the embryos are transferred to either solid or liquid MS
medium supplemented with 500 mg/L carbenicillin or 300 mg/L cefotaxime to
kill the agrobacteria. The liquid medium is used to moisten the sterile
filter paper. The embryos are incubated during 4 weeks at 25° C.,
under 150 μmol m-2 sec-1 and 12 hours photoperiod. Once the
seedlings produce roots, they are transferred to sterile metromix soil.
The medium of the in vitro plants is washed off before transferring the
plants to soil. The plants are kept under a plastic cover for 1 week to
favor the acclimatization process. Then the plants are transferred to a
growth room where they are incubated at 25° C., under 150 μmol
m-2 sec-1 light intensity and 12 hours photoperiod for about 80
days.

[0206]The transgenic plants are then screened for their improved drought,
salt and/or cold tolerance according to the screening method described in
Example 7 to demonstrate that transgene expression confers stress
tolerance.

[0208]The method of plant transformation described herein is also
applicable to Brassica and other crops. Seeds of canola are surface
sterilized with 70% ethanol for 4 minutes at room temperature with
continuous shaking, followed by 20% (v/v) Clorox supplemented with 0.05%
(v/v) Tween for 20 minutes, at room temperature with continuous shaking.
Then, the seeds are rinsed 4 times with distilled water and placed on
moistened sterile filter paper in a Petri dish at room temperature for 18
hours. Then the seed coats are removed and the seeds are air dried
overnight in a half-open sterile Petri dish. During this period, the
seeds lose approx. 85% of its water content. The seeds are then stored at
room temperature in a sealed Petri dish until further use. DNA constructs
and embryo imbibition are as described in Example 10. Samples of the
primarily transgenic plants (T0) are analyzed by PCR to confirm the
presence of T-DNA. These results are confirmed by Southern hybridization
in which DNA is electrophoresed on a 1% agarose gel and transferred to a
positively charged nylon membrane (Roche Diagnostics). The PCR DIG Probe
Synthesis Kit (Roche Diagnostics) is used to prepare a
digoxigenin-labelled probe by PCR, and used as recommended by the
manufacturer.

[0209]The transgenic plants are then screened for their improved stress
tolerance according to the screening method described in Example 7 to
demonstrate that transgene expression confers drought tolerance.

[0211]Transformation of maize (Zea Mays L.) is performed with the method
described by Ishida et al. 1996. Nature Biotch 14745-50. Immature embryos
are co-cultivated with Agrobacterium tumefaciens that carry "super
binary" vectors, and transgenic plants are recovered through
organogenesis. This procedure provides a transformation efficiency of
between 2.5% and 20%. The transgenic plants are then screened for their
improved drought, salt and/or cold tolerance according to the screening
method described in Example 7 to demonstrate that transgene expression
confers stress tolerance.

[0213]Transformation of wheat is performed with the method described by
Ishida et al. 1996 Nature Biotch. 14745-50. Immature embryos are
co-cultivated with Agrobacterium tumefaciens that carry "super binary"
vectors, and transgenic plants are recovered through organogenesis. This
procedure provides a transformation efficiency between 2.5% and 20%. The
transgenic plants are then screened for their improved stress tolerance
according to the screening method described in Example 7 to demonstrate
that transgene expression confers drought tolerance.

Example 14

Identification of Homologous and Heterologous Genes

[0214]Gene sequences can be used to identify homologous or heterologous
genes from cDNA or genomic libraries, Homologous genes (e.g. full-length
cDNA clones) can be isolated via nucleic acid hybridization using for
example cDNA libraries. Depending on the abundance of the gene of
interest, 100,000 up to 1,000,000 recombinant bacteriophages are plated
and transferred to nylon membranes. After denaturation with alkali, DNA
is immobilized on the membrane by e.g. UV cross linking. Hybridization is
carried out at high stringency conditions. In aqueous solution
hybridization and washing is performed at an ionic strength of 1 M NaCl
and a temperature of 68° C. Hybridization probes are generated by
e.g. radioactive (32P) nick transcription labeling (High Prime,
Roche, Mannheim, Germany). Signals are detected by autoradiography.

[0215]Partially homologous or heterologous genes that are related but not
identical can be identified in a manner analogous to the above-described
procedure using low stringency hybridization and washing conditions. For
aqueous hybridization, the ionic strength is normally kept at 1 M NaCl
while the temperature is progressively lowered from 68 to 42° C.

[0216]Isolation of gene sequences with homologies (or sequence
identity/similarity) only in a distinct domain of (for example 10-20
amino acids) can be carried out by using synthetic radio labeled
oligonucleotide probes. Radio labeled oligonucleotides are prepared by
phosphorylation of the 5-prime end of two complementary oligonucleotides
with T4 polynucleotide kinase. The complementary oligonucleotides are
annealed and ligated to form concatemers. The double stranded concatemers
are than radiolabeled by, for example, nick transcription. Hybridization
is normally performed at low stringency conditions using high
oligonucleotide concentrations.

Oligonucleotide hybridization solution;

6×SSC

[0217]0.01 M sodium phosphate

1 mM EDTA (pH 8)

0.5% SDS

[0218]100 μg/ml denatured salmon sperm DNA0.1% nonfat dried milk

[0219]During hybridization, temperature is lowered stepwise to
5-10° C. below the estimated oligonucleotide Tm or down to room
temperature followed by washing steps and autoradiography. Washing is
performed with low stringency such as 3 washing steps using 4×SSC.
Further details are described by Sambrook, J. et al. (1989), "Molecular
Cloning: A Laboratory Manual", Cold Spring Harbor Laboratory Press or
Ausubel, F. M. et al. (1994) "Current Protocols in Molecular Biology",
John Wiley & Sons.

Example 15

Identification of Homologous Genes by Screening Expression Libraries with
Antibodies

[0220]cDNA clones can be used to produce recombinant protein for example
in E. coli (e.g. Qiagen QIAexpress pQE system). Recombinant proteins are
then normally affinity purified via Ni-NTA affinity chromatography
(Qiagen). Recombinant proteins are then used to produce specific
antibodies for example by using standard techniques for rabbit
immunization. Antibodies are affinity purified using a Ni-NTA column
saturated with the recombinant antigen as described by Gu et al., 1994
BioTechniques 17:257-262. The antibody can than be used to screen
expression cDNA libraries to identify homologous or heterologous genes
via an immunological screening (Sambrook, J. et al. (1989), "Molecular
Cloning: A Laboratory Manual", Cold Spring Harbor Laboratory Press or
Ausubel, F. M. et al. (1994) "Current Protocols in Molecular Biology",
John Wiley & Sons).

Example 16

In Vivo Mutagenesis

[0221]In vivo mutagenesis of microorganisms can be performed by passage of
plasmid (or other vector) DNA through E. coli or other microorganisms
(e.g. Bacillus spp. or yeasts such as Saccharomyces cerevisiae) which are
impaired in their capabilities to maintain the integrity of their genetic
information. Typical mutator strains have mutations in the genes for the
DNA repair system (e.g., mutHLS, mutD, mutT, etc.; for reference, see
Rupp, W. D. (1996) DNA repair mechanisms, in: Escherichia coli and
Salmonella, p. 2277-2294, ASM: Washington.) Such strains are well known
to those skilled in the art. The use of such strains is illustrated, for
example, in Greener, A. and Callahan, M. (1994) Strategies 7:32-34.
Transfer of mutated DNA molecules into plants is preferably done after
selection and testing in microorganisms. Transgenic plants are generated
according to various examples within the exemplification of this
document.

Example 17

In Vitro Analysis of the Function of Physcomitrella Genes in Transgenic
Organisms

[0222]The determination of activities and kinetic parameters of enzymes is
well established in the art. Experiments to determine the activity of any
given altered enzyme must be tailored to the specific activity of the
wild-type enzyme, which is well within the ability of one skilled in the
art, Overviews about enzymes in general, as well as specific details
concerning structure, kinetics, principles, methods, applications and
examples for the determination of many enzyme activities may be found,
for example, in the following references: Dixon, M., and Webb, E. C.,
(1979) Enzymes, Longmans: London; Fersht, (1985) Enzyme Structure and
Mechanism. Freeman: New York; Walsh, (1979) Enzymatic Reaction
Mechanisms. Freeman: San Francisco; Price, N. C., Stevens, L. (1982)
Fundamentals of Enzymology. Oxford Univ. Press: Oxford; Boyer, P. D., ed.
(1983) The Enzymes, 3 rd ed. Academic Press: New York; Bisswanger, H.,
(1994) Enzymkinetik, 2nd ed. VCH: Weinheim (ISBN 3527300325);
Bergmeyer, H. U., Bergmeyer, J., Graβl, M., eds. (1983-1986) Methods
of Enzymatic Analysis, 3rd ed., vol, I-XII, Verlag Chemie: Weinheim;
and Ullmann's Encyclopedia of Industrial Chemistry (1987) vol. A9,
Enzymes. VCH: Weinheim, p. 352-363.

[0223]The activity of proteins which bind to DNA can be measured by
several well-established methods, such as DNA band-shift assays (also
called gel retardation assays). The effect of such proteins on the
expression of other molecules can be measured using reporter gene assays
(such as that described in Kolmar, H. et al. (1995) EMBO J. 14:3895-3904
and references cited therein). Reporter gene test systems are well known
and established for applications in both pro- and eukaryotic cells, using
enzymes such as β-galactosidase, green fluorescent protein, and
several others.

[0224]The determination of activity of membrane-transport proteins can be
performed according to techniques such as those described in Gennis, R.
B. Pores, Channels and Transporters, in Biomembranes, Molecular Structure
and Function, pp. 85-137, 199-234 and 270-322, Springer: Heidelberg
(1989).

Example 18

Purification of the Desired Product from Transformed Organisms

[0225]Recovery of the desired product from plant material (i.e.,
Physcomitrella patents or Arabidopsis thaliana), fungi, algae, ciliates,
C. glutamicum cells, or other bacterial cells transformed with the
nucleic acid sequences described herein, or the supernatant of the
above-described cultures can be performed by various methods well known
in the art. If the desired product is not secreted from the cells, can be
harvested from the culture by low-speed centrifugation, the cells can be
lysed by standard techniques, such as mechanical force or sonification.
Organs of plants can be separated mechanically from other tissue or
organs. Following homogenization cellular debris is removed by
centrifugation, and the supernatant fraction containing the soluble
proteins is retained for further purification of the desired compound. If
the product is secreted from desired cells, then the cells are removed
from the culture by low-speed centrifugation, and the supernate fraction
is retained for further purification.

[0226]The supernatant fraction from either purification method is
subjected to chromatography with a suitable resin, in which the desired
molecule is either retained on a chromatography resin while many of the
impurities in the sample are not, or where the impurities are retained by
the resin while the sample is not. Such chromatography steps may be
repeated as necessary, using the same or different chromatography resins.
One skilled in the art would be well-versed in the selection of
appropriate chromatography resins and in their most efficacious
application for a particular molecule to be purified. The purified
product may be concentrated by filtration or ultrafiltration, and stored
at a temperature at which the stability of the product is maximized.